A full PADI Divemaster course, with all the practical and theory components, but you will be with us until you have gained over 100 dives. Ideal for candidates that want to complete the course, are happy to be focused full time, and are keen to be employed as soon as graduation.
Our course is designed to produce employable DM's, with a strong industry reputation. If we do employ Divemasters we recruit from in-house.
As part of our internship, you gain your full Medical Clearance and OSH certification, and you will be mentored by our experienced crew. There will be theory and classroom work, but most days you will be at sea, and diving!
$3900 includes all your equipment, air fills, and boat trips, including PADI materials.
$3400 if you have your own equipment, and we are just supplying you with tanks and weights.
You must complete the theory online with PADI prior to your first day with us.
We do not role play. You will be assisting on actual courses, and gaining genuine and not staged experiences!
Our Internship has all the criteria and inclusions as the Divemaster course, but there is no time frame, and you will be with us until your dives are completed. Especially good if you have under 50 dives to start with, as you are guaranteed to get to 100 with us!
As part of your Internship you will need to obtain Medical Clearance, and this involves a full dive medical by a designated dive doctor.
You will need to produce the statement declaring you Fit to Dive.
The medical will cost $250 and the application fee is $97.
A hardworking but fun outlook on life!
Once you are a Divemaster, and have 100 dives under your belt, it is a simple step to obtain your COC, and a paid job doing what you love!
To be legally able to work, you will need an OSH certification (Occupational Safety and Health) and a Certificate of Competency to be fully employed in New Zealand as a dive professional.
All your equipment, and air fills, all trips out to the wrecks and the Poor Knights, all transfers and pool costs, all the knowledge you can absorb from our talented team!
We will pay your initial application and professional fees to PADI. Annual renewal is your responsibility.
We give you the required PADI materials from PADI. A DM CrewPak has everything you need to succeed.
We tailor these course to suit you, so just drop us a line and we will work with you to get you diving as a professional in the industry.
Email us, pay a deposit, we give you eLearning code, you start the study, and then you arrive here to dive!
No problems, we can get a DMPrep Package of dives in for you - two dives a day, at half price on any of our boats.


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Over 30 People Injured in Traffic Accident in Hong Kong - Re ..
Over 30 People Injured In Traffic Accident In Hong Kong - Reports
Sehar Sial 2 months ago
More than 30 people were injured in an accident involving a double-decker in Hong Kong on Saturday and at least 12 of them were taken to hospital, the South China Morning Post reported, citing the fire service
BEIJING (Pakistan Point News / Sputnik - 16th November, 2019) More than 30 people were injured in an accident involving a double-decker in Hong Kong on Saturday and at least 12 of them were taken to hospital, the South China Morning Post reported, citing the fire service.
The double-decker bus, which was heading from the city's central district to the Tai Po district, crashed into a road barrier before entering a tunnel.
According to a preliminary investigation, the driver of the vehicle lost control for an unknown reason. Firemen and doctors arrived at the scene, they were able to quickly help all passengers get out of the bus. Three people received serious injuries, according to firemen.
Paris Police Ban Yellow Vest Rallies After Protesters Clash With Offic ..
Pro-Independence Protesters Storm Barcelona Railway Station
Sehar Sial
Over 50 People Injured in Clashes in Bei ..
Trudeau, Erdogan Agree on Need to De-Esc ..
UN Human Rights Experts to Pursue Dialog ..

redpajama_set_name: RedPajamaCommonCrawl


text:
La Sinagoga d'Asmara (en hebreu: בית הכנסת של אסמרה) és l'únic edifici religiós supervivent de la comunitat jueva del país africà d'Eritrea. Inclou un cementiri jueu, aules i un santuari principal. Tots els aspectes de la sinagoga, avui són atesos per Samuel Cohen, un nadiu d'Asmara que va romandre al país per tenir-ne cura.

Referències

Eritrea
Sinagogues

redpajama_set_name: RedPajamaWikipedia


text:
French ambassador: Smooth transition between presidency stints in common interest powered by
Wallonia's red card for CETA is a chance for the EU to be a beacon to the world
DISCLAIMER: All opinions in this column reflect the views of the author(s), not of EURACTIV Media network.
By Paul de Clerck | Friends of the Earth Europe
CETA and TTIP have generated unprecedented levels of public opposition in the EU. [Friends of the Earth Europe/Flickr]
Comments Print Email Facebook Twitter LinkedIn WhatsApp
We are at a crossroads for international trade policy. The failure of TTIP and CETA would pave the way for a fairer and more democratic world, argues Paul de Clerck.
Paul de Clerck is the economic justice programme coordinator for Friends of the Earth Europe.
The European Union, the world's biggest trading bloc, is, for the second time in a matter of months, on the verge of seeing one of its mega trade deals fall into disarray.
If the objections of several regions in Belgium are upheld, the signing of the Comprehensive Economic and Trade Agreement (CETA) with Canada planned for next week will most likely be cancelled. This follows the near collapse of TTIP, the EU's proposed agreement with the US. Rather than despairing at the state of the EU, we should see how these developments provide a unique opportunity for the EU to take the lead in shaping new trade regimes for the future that are beneficial for people and the environment.
The opposition to CETA and TTIP has been unprecedented in the history of the EU. Concerns have been expressed by millions of people across the continent, including lawyers, academics, political parties, local authorities and virtually all sectors of civil society. Many governments have also expressed reservations on CETA. Only the Walloons, however, had the guts to show it the red card.
Wallonia, the Belgian region taking the lead in its steadfast opposition to CETA, is doing us all a favour. For a start, they are exercising their democratic right to raise concerns about a proposed EU deal. One of the most important issues raised by Wallonia is how CETA would give power to foreign investors to sue governments for huge compensation if democratically agreed decisions to protect citizens or the environment interfere with their profits. Wallonia faces not only substantial financial liabilities, but also a serious impairment to its democratic decision-making powers. It is only normal that the region should have its say in this matter.
Secondly, the Walloon parliament has actually gone further than most other governments and parliaments in the EU: it seriously looked at the text of CETA and debated the pros and cons of such an agreement before taking a principled position. Sadly, similar criticism of CETA in Germany led to the drafting of an additional declaration to the agreement that acts as a palliative without addressing any of the issues raised, especially when it comes to the controversial investment chapter. Again, the Walloons are right to not be fooled by this PR exercise and remain steadfast in their opposition as long as their concerns are not answered properly.
The predictable scare-mongering by the political elite has already begun. They claim that if CETA fails then the EU would no longer be able to make such trade agreements in the future. But what if this was a good thing?
Negotiated under former prime minister Harper, one of Canada's most conservative leaders in decades, the claims that CETA is the most progressive trade agreement ever made is total nonsense. At best, the agreement will just shift goods – and jobs – from one continent to another. In all likelihood, it will shift the balance of power away from democracy towards big corporations and traders. The intense debate of the last three years on TTIP and CETA shows these agreements do not stand up to the slightest bit of scrutiny. Nor do they deal with any of the pressing global issues. Yet the impacts of increased liberalisation and globalisation at all costs are open for all to see: rising inequality, unabated damage to our planet and increasing disenchantment with mainstream politics, as well as the EU project. So it should be no surprise to see citizens standing up and saying enough is enough.
It is time to develop a new trade policy for the EU and national governments that is publicly acceptable, transparent and fit for the future. A new trade regime should contribute to cutting inequalities, eliminating tax evasion, halting the collapse of biodiversity and preventing climate change. It should strengthen our democracies by ensuring public safeguards and laws are agreed openly and away from the hands of trade negotiators and big business.
Citizens also have a keen eye for fairness. Our new trade deals should not just be about protecting our own interests but also allowing other regions in the world to develop sustainably and fairly. The EU can and should take the lead in shaping this.
If it takes the Walloon government to throw a spanner in the CETA works and create the political space for an open discussion on what Europe really needs, we fully support them. Wallonia will not just protect its 3 million inhabitants. It will also defend the interests of the vast majority of the EU's 500 million citizens. Our European decision-makers should grasp this opportunity. Now is the chance for the EU to light the way ahead, reclaim social democracy and lead on establishing an economic system that contributes to a more equitable and sustainable world.
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3Poland says Germany wants to turn EU into 'Fourth Reich'
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6Pundits: Foul play on the Bulgarian gas market profits Russia
7Code Red for Humanity - or Green Light for more climate destruction?
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10What to expect in EU policymaking in 2022
5 responses to "Wallonia's red card for CETA is a chance for the EU to be a beacon to the world"
Victor Mikov says:
This matter warrants a referendum!
peter wraight says:
CETA and other trade deals for big business are just that, they are not for the average person and bring no personal benefits. I am the average Canadian and will get no benefit from this deal either and would urge everyone to say no to the corporate elite & big business.
CETA and TTIP have generated unprecedented levels of public opposition in the EU. The people aren't stupid as the greedy suits would like them to be.
simon.ward@increment.co.uk says:
Life's complicated. What is wrong with cheaper food for the poorest in society?
an european says:
Give Wallonians their belge franc back and purge barriers on it !


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Q: Writing PHP in Notepad++, and using Spanish letters? I don't get it - what encoding should I use? I've been using ANSI, but when I save the document, Spanish letters are converted to illegible markings.
Any suggestions?

A: you should use UTF-8 without BOM

A: In general, for almost any document, you should use UTF-8 without a BOM. (The exception is documents targeting most Asian countries which are usually more efficiently stored in UTF-16).
You also need to make sure that:

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"Happiness is like manna; it is to be gathered…and enjoyed every day. We have to go out of ourselves…to gather it, since it has rained down from a Heaven at our very door." ~ Tryon Edwards (American theologian 1809-1894).
Yesterday's blog about fickle Mother Nature made me think of a film that is currently playing in theatres across the country. The title of it, Cloudy with a Chance of Meatballs 2, sounds like a description of our country's political and economic landscape at this moment, but I assure you, the storyline is quite different.
The book Cloudy with a Chance of Meatballs is manna for the soul. I must have read it to my son a hundred times or more, over the course of his growing up. It was a favorite and we returned to it time and time again, especially when we wanted to end the day on a positive note. I could probably recite the text by heart.
The illustrations in the book made us laugh – images of Mother Nature releasing storms of mashed potatoes, meatballs and spaghetti. There were memorable pictures of torrential downpours of salt and pepper, people being flattened by flying hamburgers.
Despite the chaos, the governing body in the town of Chew and Swallow didn't shut down. The plow trucks were out removing piles of peas and carrots blocking the streets and making order out of mess. They were working hard to solve the problems and change the landscape from rotten eggs clogging progress to a more sunny side up environment.
Now that I have been involved in writing and publishing, I see the book as a great example of how an author, Judi Barrett, used her imagination to create a wonderfully entertaining story for both children and adults.
Ahh… the days of reading bed time stories. My son, Marc, is now twenty-five years old, and a filmmaker specializing in visual effects. He also works as a film colorist. A perfect way to spend a holiday evening would be to watch this film and escape to the fictional town of Chew and Swallow together where we can look in on his world, and he will have fun providing feedback about the movie.
I think I'll put the DVD on my shopping list for Christmas…..sshhhhh…don't tell Marc.
Here is where to purchase the movie if you can't catch it at the theatre. http://www.imdb.com/title/tt1985966/ .


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US6020004A - Biodegradable microparticles for the sustained delivery of therapeutic drugs - Google Patents
Biodegradable microparticles for the sustained delivery of therapeutic drugs Download PDF
method according
ob protein
Subodh Shah
Amgen Inc
1997-04-17 Application filed by Amgen Inc filed Critical Amgen Inc
1997-04-17 Assigned to AMGEN INC. reassignment AMGEN INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHAH, SUBODH
1998-04-17 Priority claimed from PT98918424T external-priority patent/PT975334E/en
1998-04-17 Priority claimed from SI9830547T external-priority patent/SI0975334T1/en
A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
A61K9/1682—Processes
A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
Y10T428/00—Stock material or miscellaneous articles
Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
The present invention relates to improved methods of making polymeric microparticles containing a variety of active ingredients, e.g. protein drugs. In addition, the present invention relates to using the above active protein containing polymeric microparticles to prepare compositions for the sustained delivery of the therapeutics.
The present invention generally relates to improved methods of making biodegradable polymeric microparticles containing an active ingredient. In addition, the present invention relates to use of said microparticles to prepare compositions for the sustained delivery of therapeutics.
Due to recent advances in genetic and cell engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of production in large amounts for pharmaceutical applications. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth differentiation factor (MGDF), osteoprotegerin (OPG), glial cell line derived neurotrophic factor (GDNF) and obesity protein (OB protein). OB protein may also be referred to herein as leptin.
Because these proteins generally have short in vivo half-lives and negligible oral bioavailability, they are typically administered by frequent injection, thus posing a significant physical burden on the patient and associated administrative costs. As such, there is currently a great deal of interest in developing and evaluating sustained-release formulations. Effective sustained-release formulations can provide a means of controlling blood levels of the active ingredient, and also provide greater efficacy, safety, patient convenience and patient compliance. Unfortunately, the instability of most proteins (e.g. denaturation and loss of bioactivity upon exposure to heat, organic solvents, etc.) has greatly limited the development and evaluation of sustained-release formulations.
Attempts to develop sustained-release formulations have included the use of a variety of biodegradable and non-biodegradable polymer (e.g. poly(lactide-co-glycolide)) microparticles containing the active ingredient (see e.g., Wise et al., Contraception, 8:227-234 (1973); and Hutchinson et al., Biochem. Soc. Trans., 13:520-523 (1985)), and a variety of techniques are known by which active agents, e.g. proteins, can be incorporated into polymeric microspheres (see e.g., U.S. Pat. No. 4,675,189 and references cited therein).
One such technique is spray-drying, wherein the polymer and active ingredient are mixed together in a solvent for the polymer, and then the solvent is evaporated by spraying the solution, leaving polymeric droplets containing the active ingredient. For a detailed review of spray drying see e.g. Masters, K., "Spray Drying Handbooks" (John Wiley & Sons, eds., New York 1984). Although the spray drying technique has proven useful in certain instances, it still suffers from the fact that biologically active proteins are often denatured due to contact with the organic polymer and solvent, or due to the heat generated during the spray drying processes.
Another technique which can be used to form microspheres is solvent evaporation. Solvent evaporation involves the dissolving of the polymer in an organic solvent which contains either dissolved or dispersed active ingredient. The polymer/active ingredient mixture is then added to an agitated continuous phase which is typically aqueous. Emulsifiers are included in the aqueous phase to stabilize the oil-in-water emulsion. The organic solvent is then evaporated over a period of several hours or more, thereby depositing the polymer around the core material. For a complete review of the solvent evaporation procedure see e.g. U.S. Pat. No. 4,389,330 (and references cited therein). As with the spray drying technique, solvent evaporation techniques have proven useful in certain instances. However, the technique is often not preferred because active ingredient is often lost during the solvent extraction process. This is because the process involves emulsification into an aqueous phase, and a water soluble drug will often rapidly partition from the more hydrophobic polymer-solution phase into the aqueous surroundings.
Yet another technique which can be used to form microspheres is phase separation, which involves the formation of a water-in-oil emulsion or oil in water emulsion. The polymer is precipitated from the continuous phase onto the active agent by a change in temperature, pH, ionic strength or the addition of precipitants. For a review of phase separation techniques, see e.g. U.S. Pat. No. 4,675,800 (and references cited therein). Again, this process suffers primarily from loss of active ingredient due to denaturation.
The release characteristics for the active ingredient from microparticles prepared by methods such as those described above may be continuous or discontinuous, and in some cases, the initial level of active ingredient release is too high or too low. Thus, various additives are often utilized in an attempt to control the release of active ingredient (see e.g., EP 0 761 211 A1, published Mar. 12, 1997).
To avoid the denaturation of protein and other fragile biological molecules which occurs upon spray drying, solvent evaporation or phase separation by classical techniques, the emulsion of polymers and active ingredient can be atomized into frozen nonsolvent overlayed with liquified gas such as nitrogen to form particles, and then extracted at very low temperatures. The extremely low processing temperatures may preserve the activity and integrity of the fragile biological molecules such as proteins. However, the method leads to poor loading efficiencies and yields, resulting in the loss of precious biological material, and is cumbersome, difficult and expensive to implement at the large scales required for commercial production.
Clearly the need still exists for an improved method for preparing polymeric microparticles containing an active ingredient which is simple, inexpensive, versatile, and, most importantly, which protects against loss of protein activity and which provides for high loading efficiencies and yields, thereby allowing for more consistent active ingredient release over an extended period of time.
As fully described below, the present invention provides an improved method for preparing polymeric microparticles containing an active ingredient through unique utilization of direct lyophilization of emulsion or suspension. This improved method provides several significant advantages over the processes described in the art, including, for example, 1) ease of manufacture of the active ingredient loaded microparticles (i.e. fewer and less cumbersome steps); and 2) the provision of sustained release formulations that maintain the activity and integrity of the active ingredient during release, thus providing for controlled release of active ingredient over an extended period of time. Additionally, the processes of the present invention provide the advantages of versatility as relates to the class of polymers and/or active ingredient which may be utilized, as well as attainment of higher yields, high loading, and higher loading efficiencies.
Accordingly, one aspect of the present invention relates to a new and improved process for preparing a composition comprising an active ingredient contained within polymeric microparticles, wherein a mixture of the active ingredient and the polymer are dispersed within a continuous phase, the resulting dispersion is frozen, and the water and organic solvents removed from the dispersion by lyophilization. Importantly, the present process is more refined and simpler than those described in the art, and the activity and integrity of the active ingredient is maintained throughout the process.
The present process can be generally described as comprising the steps of: (a) preparing a polymeric solution; (b) adding an active ingredient to produce a mixture; (c) dispersing said mixture within a continuous phase to produce a dispersion; (d) adding an excipient to said dispersion; (e) freezing said dispersion to produce a frozen mixture; and (f) lyophilizing said frozen mixture to produce the desired active ingredient containing microparticles. Alternatively, step (b) can be omitted and "blank" microparticles prepared, onto which active ingredient is then loaded by suspending the blank microparticles in active ingredient solution.
A second aspect of the present invention is a pharmaceutical composition for the sustained-release of an active ingredient comprising a biologically active ingredient contained within polymeric microparticles, or, alternatively, a biologically active ingredient loaded onto the polymeric microparticles. Importantly, the sustained-release compositions of the present invention maintain the activity and integrity of the active ingredient during encapsulation and release, which helps to provide for longer periods of consistent release.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic of the process of the present invention for making the active ingredient containing microparticles.
FIG. 2 is a plot depicting the in vitro release of OB protein (leptin) from OB protein loaded microparticles. % OB protein released (OB protein concentration having been determined by UV spectrophotometer at 280 nm) is plotted vs. time (days).
FIG. 3 shows the circular dichroism (CD) data comparing OB protein released from OB protein loaded microparticles (in vitro) on day 7 (solid line) vs. a control sample of OB protein in formulation buffer (dashed line). CD spectra were obtained with a Jasco J-720 spectrapolarimeter (Japan Spectroscopic Co., Tokyo, Japan). The samples (3.5 μM as determined by A280) were analyzed at 22° C. with a cell path length of 0.1 cm.
FIG. 4 shows the high performance liquid chromatography (HPLC) data for OB protein released from OB protein loaded microparticles (in vitro) at various time points (0 to 168 hours). Analytical Size Exclusion Chromatography (SEC) was performed with a TosoHaas G2000 SW column (Montgomery, Pa.) using a Waters HPLC (Milford, MA) with 20 mM sodium phosphate, 125 mM NaCl, pH 7.4 at 0.8 mL/min. Absorbance at 280 nm is plotted vs. run time (minutes).
FIG. 5 is a picture of an SDS-PAGE gel (2-20% Tris-glycine gel (Novex, San Diego, Calif.)) containing the following samples: lane 1: OB protein standard; lane 2: molecular weight standards; lanes 3-9: OB protein released from OB protein loaded microparticles (in vitro) at 2 hours, 24 hours, 68 hours, 92 hours, 116 hours, 140 hours, and 168 hours, respectively; lane 10, molecular weight standards. Samples were diluted with nonreducing SDS buffer, heated at 100° C. for five minutes and 1 μg of protein loaded into each well. The gels were stained with Coomassie Blue R-250.
FIG. 6 shows in vivo bioactivity of OB protein (leptin) loaded microparticles in normal mice, in terms of % body weight loss relative to buffer control for a 7 day period. Buffer control (-*-) is plotted vs. leptin injected @ 10 mg/kg daily (--) vs. leptin injected @ 50 mg/kg on day 0 only (-x-) vs. leptin loaded microparticles injected @ 50 mg/kg on day 0 only (- -) vs. control microparticles injected on day 0 only (-∘-).
FIG. 7 is a plot depicting the in vitro release of BDNF from microparticles onto which BDNF had been absorbed. % BDNF released (BDNF protein concentration having been determined by UV spectrophotometer at 280 nm) is plotted vs. time (days).
Polymers may be selected from the group consisting of biocompatible and/or biodegradable polymer. As defined herein, biodegradable means that the composition will erode or degrade in vivo to form smaller chemical species. Degradation may occur, for example, by enzymatic, chemical or physical processes. Suitable biodegradable polymers contemplated for use in the present invention include poly(lactide)s, poly(glycolide)s, poly(lactic acid)s, poly(glycolic acid)s, polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymers thereof.
The range of molecular weights contemplated for the polymers to be used in the present processes can be readily determined by a person skilled in the art based upon such factors the desired polymer degradation rate. Typically, the range of molecular weight will be 2000 to 2,000,000 Daltons. Almost any type of polymer can be used provided the appropriate solvent and non-solvent are found.
The term "PLGA " as used herein is intended to refer to a polymer of lactic acid alone, a polymer of glycolic acid alone, a mixture of such polymers, a copolymer of glycolic acid and lactic acid, a mixture of such copolymers, or a mixture of such polymers and copolymers. Preferably, the biodegradable polymer will be poly lactide-co-glycolide (PLGA).
Unless otherwise noted, the term microparticles can be used to encompass microparticles, microspheres, and microcapsules. Active agents to be incorporated into the microparticles are synthetic or natural compounds which demonstrate a biological effect when introduced into a living creature. Contemplated active agents include peptides, small molecules, carbohydrates, nucleic acids, lipids, and proteins. Proteins contemplated for use include potent cytokines, including various hematopoietic factors such as G-CSF, GM-CSF, M-CSF, MGDF, the interferons (alpha, beta, and gamma), interferon consensus, the interleukins (1-12), erythropoietin (EPO), fibroblast growth factor, TNF, TNFbp, IL-1ra, stem cell factor, nerve growth factor, GDNF, BDNF, NT3, platelet-derived growth factor, and tumor growth factor (alpha, beta), osteoprotegerin (OPG), and OB protein.
Also contemplated for incorporation into the compositions of the present invention are derivatives, fusion proteins, conjugates, analogs or modified forms of the natural active ingredients. Chemical modification of biologically active proteins has been found to provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. For example, U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979, discloses conjugation of water-soluble polypeptides such as enzymes and insulin to polyethylene glycol (PEG); see also WO 87/00056, published Jan. 15, 1987.
Another type of chemical modification contemplated for the active ingredients of the present invention is succinylation. The properties of various succinylated proteins are described in Holcenberg et al., J. Biol. Chem, 250:4165-4170 (1975), and WO 88/01511 (and references cited therein), published Mar. 10, 1988.
The present OB proteins used are preferably those with amino acid sequence of natural human OB protein; see Zhang et al., Nature 3:425-432 (1994); see also, the Correction at Nature 374: 479 (1995), optionally with an N-terminal methionyl residue incident to bacterial expression is used. (See, Materials and Methods, infra). PCT publication No. WO 96/05309, published Feb. 22, 1996, entitled, "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses Thereof" fully sets forth OB protein and related compositions and methods, and is herein incorporated by reference. An amino acid sequence for human OB protein is set forth at WO 96/05309 Seq. ID Nos. 4 and 6 (at pages 172 and 174 of that publication), and the first amino acid residue of the mature protein is at position 22 and is a valine residue. The mature protein is 146 residues (or 145 if the glutamine at position 49 is absent, Seq. ID No. 4).
Specific OB protein derivatives contemplated for use in the present invention include Fc-OB protein fusions, succinylated-OB protein, and zinc derivatized OB protein. It is desirable to have such OB protein containing sustained-release compositions as such compositions could serve to enhance the effectiveness of either exogenously administered or endogenous OB protein, or could be used, for example, to reduce or eliminate the need for exogenous OB protein administration.
In general, an aqueous solution, a suspension, or a solid form of the active agent can be admixed with the organic solvent containing the polymer. When an aqueous solution of active ingredient is used, polymer:active ingredient emulsions will be formed and used to prepare microparticles. When a suspension or solid form of active ingredient is used, polymer:active ingredient suspensions are formed and used to prepare the microparticles.
The principal embodiment of the method for making the protein loaded microparticles comprises: (a) dissolving a polymer in an organic solvent to produce a polymeric solution; (b) adding an active ingredient in a form selected from the group consisting of an aqueous solution, a suspension, and a powder to said polymeric solution to produce a active ingredient-polymer mixture comprising a first emulsion or suspension; (c) dispersing said first emulsion or suspension within a continuous phase to produce a dispersion; (d) adding an excipient to said dispersion to produce a final dispersion; (e) freezing said final dispersion; and (f) lyophilizing said frozen final dispersion to remove different solvents (aqueous and organic) to produce the desired protein loaded microparticles. The process is shown schematically in FIG. 1. As depicted in FIG. 1, step c) can alternatively comprise diluting said first emulsion or suspension with a polymer non-solvent. Additionally, step a) can comprise active ingredient being dissolved directly in the organic polymer solution to form a homogeneous first mixture.
The solvent to be used for dissolving the PLGA in step a) of the present processes includes, for example, chloroform, ethyl acetate, methylene chloride, acetonitrile, THF and acetone. In one embodiment of the present invention, the solvent to be used is chloroform. Non-solvents contemplated for use in step c) include water, hexane, ethanol, methanol, and carbon tetrachloride.
The polymer concentrations contemplated for use in the processes of the present invention are in the range of 5-70 gm/100 mL. In the embodiments of the present invention which utilize PLGA, the polymer concentration will preferably be in the range of 10-20 gm/100 mL.
The protein concentrations contemplated for use in the processes of the present invention are in the range of 0-300 mg/mL when in solution or suspension, or equivalent solid protein. In the embodiments of the present invention which utilize OB protein, the protein concentration is preferably 100 mg/mL.
For the emulsions produced in the processes of the present invention, the organic:aqueous ratios contemplated for use are 1:1 to 12:1. In the embodiments of the present invention which utilize PLGA and OB protein, the organic:aqueous ratio will preferably be 4:1 for the first emulsion. In general, the microparticles prepared by the methods of the present invention will generally comprise 0-60% by weight of protein.
The addition of a lyophilization excipient in step d) of the process described above was found to be necessary to insure that the microparticles did not aggregate or fuse during lyophilization. One or more excipients may be added. Importantly, such excipient(s) could also be added in step b) or c) of the process. The lyophilization excipient(s) contemplated for use in the present processes include lactose, mannitol, dextran, sucrose, heparin, glycine, glucose, glutamic acid, gelatin, sorbitol, dextrose, trehalose, methocel, hydroxy ethyl cellulose, hydroxy ethyl starch, poly(ethylene glycol), poly(vinyl pyrolidone) and polyvinyl alcohol, or various combinations thereof, as well as other buffers, protein stabilizers, cryoprotectants, and cyropreservatives commonly used by those skilled in the art.
The temperatures contemplated for use in the freezing step (step e) of the present processes are in the range of -283° C. (liquid nitrogen) to -20° C. These temperatures are utilized so as to stabilize the emulsions or suspensions. The final emulsion or suspension can be frozen immediately using the temperatures described above, or can be stored prior to freezing. In one embodiment of the present invention, the final emulsion was frozen immediately and the temperature utilized for the freezing was -80° C.
The temperatures contemplated for use in step f) of the present processes are in the range of -100° C. to room temperature. Preferably, the temperature of the frozen sample of step e) will be lowered -80° C. and held for one hour prior to being connected to the vacuum system. The temperature is then raised step-wise in 5° C./hour increments to -25° C. to effect removal of the aqueous phase and any residual organic phase. The sample is then held under vacuum for 4-5 days (or whenever the vacuum gauge indicates no more vapor removal), and then the temperature raised to -5° C. for 6-8 hours before the removing the sample from the vacuum system. It is utilization of this single step, i.e., direct lyophilization of the final emulsion or suspension, which refines and simplifies the present process over previously described processes, which require multiple steps and are often cumbersome. And, importantly, this direct lyophilization provides the enhanced stability for a wide variety of active ingredient in vivo, as well as the attainment of higher loading, higher loading efficiencies, and higher yields.
Thus, the significant advantages of the present processes as compared to the processes described in the art, include for example, 1) ease of manufacture of the active ingredient loaded microparticles; 2) versatility as relates to the class of polymers and/or active ingredients which may be utilized; 3) higher yields and loading efficiencies; and 4) the provision of sustained release formulations that release active, intact active ingredient in vivo, thus providing for controlled release of active ingredient over an extended period of time (e.g. up to 180 days). As used herein the phrase "contained within" denotes a method for formulating an active ingredient into a composition useful for controlled release, over an extended period of time of the active ingredient.
In the sustained-release compositions of the present invention, an effective amount of active ingredient will be utilized. As used herein, sustained release refers to the gradual release of active ingredient from the polymer matrix, over an extended period of time. The sustained release can be continuous or discontinuous, linear or non-linear, and this can be accomplished using one or more polymer compositions, drug loadings, selection of excipients, or other modifications.
In general, comprehended by the present invention are pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. An effective amount of active ingredient is a therapeutically, prophylactically, or diagnostically effective amount, which can be readily determined by a person skilled in the art by taking into consideration such factors as body weight, age, therapeutic or prophylactic or diagnostic goal, and release rate desired.
A suspension of protein loaded microparticles prepared in accordance with the present invention is preferably administered by injection intraperitoneally, subcutantenously, or intramuscularly. However, it would be clear to one skilled in the art that other routes of delivery could also be effectively utilized using the compositions of the present invention.
The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. Example 1 describes the novel method for preparing protein loaded microparticles. OB protein (in the form of an aqueous solution) is used as an example protein and the ability of OB protein loaded microparticles to provide for sustained release of OB protein, both in vitro and in vivo is demonstrated. Example 2 demonstrates that different polymers can be used to make OB protein loaded microparticles. Example 3 demonstrates that different OB protein derivatives, as well as entirely different proteins (all in the form of an aqueous solution), can be used in the novel methods of the present invention. Example 4 demonstrates the effects of temperature on the freezing step and on the lyophilization step of the process of the present invention. Example 5 demonstrates that different organic solvents can be used to dissolve the PLGA polymers in the methods of the peasant invention. Example 6 demonstrates that a OB protein suspension, Zn/OB protein, can be used in the novel methods of the present invention. Example 7 demonstrates that a spray-dried protein, spray-dried IL-1ra, can be used in the novel methods of the present invention. Example 8 demonstrates that "blank" microparticles onto which active ingredient (e.g. BDNF) has been absorbed, can also provide for sustained release of active ingredient in vitro. Materials and methods follow.
This example describes the novel method for preparing protein loaded microparticles; specifically, the preparation of poly(D,L-lactide-co-glycolide) microspheres containing OB protein.
0.6 g of RG-502H, poly(D,L-lactide-co-glycolide) (Boehringer Ingelheim Chemicals (B.I. Chemicals), Henley Div., Montvale, N.J.) was dissolved in 4 mL of chloroform and filtered through a 0.2 μm PTFE filter. 1 mL of OB protein at 100 mg/mL in 10 mM sodium acetate, pH 4.8 (prepared as described in Materials and Methods, infra), was first sterile filtered and then gently added to the top of the polymer solution. The two layers were homogenized using a Polytron homogenizer (PT-DA3012/2T generator, Brinkman, Westbury, N.Y.) at 15,000 to 20,000 rpm for 30-45 seconds while the emulsion container was immersed in an ice bath.
The resultant first emulsion (w/o) was added to 10 mL of water while homogenizing at 15,000 rpm for 20-30 sec. To the resulting second emulsion (w/o/w), 1 mL of lyophilization excipient (100 mg/mL Glycine, 100 mg/mL Sucrose, 10 mg/mL polyvinyl-alcohol (PVA) [22,000 M.W., 88% Hydrolyzed], 10% v/v ethanol) was added and briefly homogenized to insure thorough mixing. The final emulsion under optical microscope showed 1-10 μm free flowing spheres. The final emulsion was poured into a flask and frozen at -45° C.
The temperature of the bath was then reduced in one hour to -80° C. After an hour at -80° C., the flask was connected to a vacuum system and lyophilization first carried out at -80° C. The vacuum level was monitored so that removal of organic solvent could be determined by a drop in vacuum to the system level. The temperature was then raised step-wise in 5° C./hour increments to -25° C. to effect the removal of the aqueous phase and any residual organic phase.
After 4-5 days when the vacuum gauge indicated no more vapor removal, the temperature of the bath was raised to -5° C. for 6-8 hours before removing the samples from the vacuum system. The microparticles were weighed and then stored at -20° C. until needed.
In vitro Release of OB Protein From PLGA Microrarticles
"In vitro" release kinetics of OB protein from the microparticles prepared as described above were determined by making a 20 mg/mL suspension of the particles in 20 mM sodium phosphate (or histidine), 5% Sorbitol, pH 7.4. At each time interval, the microsphere suspension was centrifuged and OB protein concentration in the supernatant was determined by UV spectrophotometer at 280 nm as well as by SEC-HPLC at 220 nm. The % OB protein released over time is depicted in FIG. 2. The integrity of the OB protein released from the PLGA microparticles was confirmed by circular dichroism (CD) (FIG. 3), HPLC (FIG. 4), in vitro bioassay and gel electrophoresis (SDS-PAGE) (FIG. 5). The CD data showed retention of secondary structure, and HPLC and gel electrophoresis showed no obvious chemical degradation or aggregation.
In vivo Bioactivity of OB Protein Loaded Microrarticles
"In vivo" bioactivity of OB protein loaded microparticles were evaluated in normal mice and rats by suspending 80-100 mg/mL microparticles in 20 mM sodium phosphate, 5% Sorbitol, pH 7.4, buffer. The suspensions were prepared an hour before subcutaneous injection by placing the microparticles and buffer on a shaker in a 5° C. cold room. All subsequent manipulations immediately prior to injection were done with refrigerated syringes and 25-gauge needles. % body weight loss relative to buffer control was determined for a 7 day period. After day 7, animals were sacrificed for histological examination of the injection site. A single injection of OB protein loaded microparticles resulted in sustained weight loss in mice for 7 days period (FIG. 6). Histological examination of the injection site revealed a localized minimal to mild inflammatory reaction, which was fully reversible with biodegradation of the microparticles over time.
This example was designed to test the effectiveness of different molecular weights of PLGA or blends in the preparation of OB protein loaded microparticles. The preparation and evaluation procedures described in Example 1 were utilized to test the various polymers listed in Table 2 below.
TABLE 2______________________________________Polymers:              Source:______________________________________RG-501H                B. I. ChemicalsRG-502H                                        B. I. ChemicalsRG-502                                          B. I. CheinicalsRG-503H                                        B. I. Chemicals(RG-501H):(RG-502H) blends*                      B. I. Chemicals(RG-501H):(PEG/PLGA)**           B. I. Chemicals______________________________________ *Polymer blends made by mixing 20:80 & 50:50 weight ratios of RG501H and RG502H **80:20 (by weight) blend of PLGA (501H) and PLGA(501H):PEG(1000) AB bloc copolymer.
Using each of the polymers listed in Table 2, protein loaded microparticles could be effectively prepared.
This example describes the preparation of microparticles containing OB protein derivatives as well as other proteins. The preparation and evaluation procedures described in Example 1 were utilized to test the various proteins listed in Table 3 below.
TABLE 3______________________________________   Molecular                 Concentration:Protein        (Daltons)              (mg/mL)          Formulation:______________________________________OB protein   16,158   50-130     10 mM NaAcO, pH 4.8-8.0                       80                                    10 mM NaAcO pH 4.8 +                                      10% Sucrose                       100                                   Lyo Buffer.sup.a                       60                                    Lipid Complexed.sup.b20 kd           ˜36,158               64-122   10 mM NaAcO, pH 4.8-8.0PEG-OBproteinSuccinylated     16,258      60-80        10 mM NaPhos, pH 7.0-8.0OB proteinG-CSF            18,798                 50-100                             10 mM NaAcO, pH 4.8-8.0                       60-100                             10 mM NaAcO, pH 4.8-8.0 +                                      5-16% Sucrose                       55                                    1 mM NaCl, 10% Trehalose,                                      pH 7.6                       60                                    Lipid Complexed.sup.bBDNF                  45-120                             100 mM NaPhos, pH 7IL-1ra          17,258                 100-200                            10 mM NaCitrate,                                      140 mM NaCl, pH 6.5TNFbp            18,278                 105               10 mM NaPhos, 2% Gly,                                      1% Sucrose, pH 7BSA                   100               10 mM NaAcO, pH______________________________________                       4.8 .sup.a Lyophilization Buffer = 10 mg/mL Glycine, 5 mg/mL Sucrose, 10 mM glutamic acid, pH 4.5. .sup.b Lipid complexed 30:1 mol. ratio DMPG or DCPG to protein in 20 mM NaAcO, pH 4.8.
With each of the proteins listed above, protein loaded microparticles were obtained, thus demonstrating the flexibility of the novel process of the present invention. And importantly, it is demonstrated that protein loaded microparticles can also be effectively prepared using different OB protein derivatives.
In this example, the effects of temperature on the freezing step and lyophilization step of the process of the present invention were evaluated. As relates to the freezing step (step 5), liquid nitrogen, -80° C. and -45° C. were tested. As relates to the lyophilization step (step 6), -80° C., -45° C., and -25° C. were tested. The procedure described in Example 1 was used to test the various temperatures and it was determined that the tested temperatures had very little effect on either step in the process.
In this example, different organic solvents were tested to dissolve the PLGA polymers in the methods of the present invention. The procedure described in Example 1 was repeated using ethyl acetate, methylene chloride. Each of the tested organic solvents were found to be effective in the methods of the present invention.
This example tested the ability of an active ingredient suspension to be used in the methods of the present invention. A 100 mg/mL, in 10 mM Tris, 50 μM Zinc chloride, pH 7.0, Zn/OB protein suspension (prepared as described in the Materials and Methods section below) was tested and evaluated as described in Example 1. It was demonstrated that the Zn/OB protein suspension could be effectively utilized to prepare microparticles according to the novel methods of the present invention.
This example tested the ability of a spray-dried protein, spray-dried IL-1ra, to be used in the methods of the present invention. A 150 mg spray-dried IL-1ra powder (prepared as described in the Materials and Methods section below) was tested and evaluated as described in Example 1. It was demonstrated that the spray-dried IL-1ra preparation could be effectively utilized to prepare microparticles according to the novel methods of the present invention.
In this example, the method described in Example 1 was modified such that 20 mM NaAcO, pH 4.8, was mixed initially with the polymer, resulting in the preparation of "blank" microparticles. 6 mg of blank microparticles was then diluted with 1 mL of BDNF (4.4 mg/mL in 0.1M sodium phosphate, pH 6.9) and the mixture incubated at 37° C. with shaking. After 2 hours, microparticles were isolated by centrifugation and the unbound protein fraction was determined by UV spectrophotometer. 1.76 mg of BDNF was bound to polymer to give 22% protein loading on the microparticles. In vitro release kinetics were then determined as described in Example 1. The % BDNF released over time is depicted in FIG. 7.
1. Preparation of Recombinant Methionyl Human OB Protein.
The present recombinant methionyl-human-OB protein may be prepared according to the above incorporated-by-reference PCT publication, WO 96/05309 at pages 151-159. For the present working examples, a human OB protein was used which has (as compared to the amino acid sequence at page 158) a lysine at position 35 instead of an arginine, and an isoleucine at position 74 instead of an isoleucine. Other recombinant human OB proteins may be prepared according to methods known generally in the art of expression of proteins using recombinant DNA technology.
2. Preparation of Spray Dried Recombinant IL-1ra.
The present spray dried recombinant IL-1ra protein was prepared by spray drying a 20 mg/mL IL-1ra solution, using a Buchi 190 mini spray dryer. The inlet and outlet temperatures during spray drying were 130° C. and 90° C., respectively. Feeding rate was 1-2 mL/min, and a spray-dried IL-1ra powder obtained.
3. Preparation of Recombinant Methionyl Succinylated Human OB Protein.
The present recombinant methionyl succinylated human OB protein was prepared by reaction of recombinant human OB protein at ˜150 mg/mL in sodium phosphate buffer at pH 7.0 with a 3-7 molar excess of succinic anhydride at 4° C. for two hours. The reaction is quenched by addition of solid hydroxyl amine to a final concentration of 0.5M and pH 8.5. The reaction mixture is then dialyzed vs. 10 mM sodium phosphate, pH 7.0 and the mono-succinylated OB protein form separated from di- and poly-succinylated OB protein forms by ion exchange chromatography.
3. Preparation of Zinc Derivatized Recombinant Methionyl Human OB Protein Suspension.
The present zinc derivatized recombinant methionyl human OB protein suspension was prepared by taking a 752 μl sample of recombinant human OB protein at 133 mg/mL in 1 mM HCl and adding 48 μl water, followed by 100 μl of 500 μM zinc chloride, followed by 100 μl of 1M TRIS, pH 8.5. There is an immediate Zn/OB protein precipitate that will rapidly fall out of solution at room temperature, but which can be readily resuspended.
While the present invention has been described in terms of certain preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed.
1. A method for making a composition comprising an active ingredient contained within polymeric microparticles, wherein a mixture of the active ingredient and the polymer are dispersed within a continuous phase, and the resulting dispersion is directly lyophilized to remove the water and organic solvents and form said microparticles.
2. A method according to claim 1 wherein said continuous phase is aqueous or organic.
3. A method according to claim 1 wherein the active ingredient-polymer mixture is obtained by dispersing an aqueous solution of the active ingredient in a second, non-aqueous phase containing the polymer, prior to addition to the continuous phase.
4. A method according to claim 1 wherein the active ingredient-polymer mixture is obtained by dissolving both components in a non-aqueous solvent prior to addition to the continuous phase.
5. A method according to claim 1 wherein the active ingredient is present as a dispersion of solid particles in a non-aqueous solution of the polymer, which is then added to the continuous phase.
6. A method according to claim 1, wherein said active ingredient is omitted from said mixture, thereby producing blank polymeric microparticles.
7. A method according to claim 6 further comprising active ingredient being loaded onto said blank polymeric microparticles by suspending said blank polymeric microparticles in active ingredient solution.
8. A method according to any of claims 1-7 wherein one or more excipients is combined with the active ingredient-polymer mixture prior to incorporation into the dispersion.
9. A method according to any of claims 1-7 wherein said continuous phase contains one or more excipients.
10. A method according to any of claims 1-7 wherein one or more excipients is mixed with the active ingredient and the same or a different excipient is present in the continuous phase.
11. A method according to claim 1 wherein said polymer is selected from the group consisting of biodegradable and/or biocompatible polymers.
12. A method according to claim 11 wherein said biodegradable polymers is selected from the group consisting of poly(lactide)s, poly(glycolide)s, poly(lactic acid)s, poly(glycolic acid)s, polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate, polyurethanes, polyacrylate, blends and copolymers thereof.
13. A method according to claim 12 wherein said polymer is poly(lactide-co-glycolide)(PLGA), and wherein said polymer is dissolved in an organic solvent selected from the group consisting of chloroform, ethyl acetate, methylene chloride, acetonitrile, THF and acetone.
14. A method according to any of claims 1-10 wherein the active ingredient is selected from the group consisting of peptides, small molecules, sugars, carbohydrates, nucleic acids, lipids, and proteins.
15. A method according to claim 14 wherein said active ingredient is a protein selected from the group consisting of G-CSF, GM-CSF, M-CSF, MGDF, the interferons (alpha, beta, and gamma), interferon consensus, the interleukins (1-12), erythropoietin (EPO), fibroblast growth factor, TNF, TNFbp, IL-1ra, stem cell factor, nerve growth factor, GDNF, BDNF, NT3, platelet-derived growth factor, tumor growth factor (alpha, beta), OPG, and OB protein; or derivatives, analogs, fusions, conjugates, or chemically modified forms thereof.
16. A method according to claim 15 wherein said protein is OB protein, or a derivative, analog, fusion, conjugate, or chemically modified form thereof.
17. A method according to claim 16 wherein said modified form of OB protein is selected from the group consisting of Fc-OB protein fusion, succinylated-OB protein, and zinc derivatized OB protein.
18. A method according to claim 15 wherein said protein is G-CSF, or a derivative, analog, fusion, conjugate, or chemically modified form thereof.
19. A method according to claim 15 wherein said protein is BDNF, or a derivative, analog, fusion, conjugate, or chemically modified form thereof.
20. A pharmaceutical composition for the sustained-release of an active ingredient, said composition produced by the method of any of claims 1-15.
21. A pharmaceutical composition comprising OB protein, or a derivative, analog, fusion, conjugate, or chemically modified form thereof contained within a polymeric microparticle.
22. A pharmaceutical composition comprising G-CSF, or a derivative, analog, fusion, conjugate, or chemically modified form thereof contained within a polymeric microparticle.
23. A pharmaceutical composition comprising BDNF, or a derivative, analog, fusion, conjugate, or chemically modified form thereof contained within a polymeric microparticle.
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Owner name: AMGEN INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SHAH, SUBODH;REEL/FRAME:008726/0458


Is paper uncitedness a function of the alphabet?
Arsenault, C., Larivière, V. (2015). Is paper uncitedness a function of the alphabet? Proceedings of the 15th International Conference of the International Society for Scientometrics and Informetrics, pp. 286-287.
Ce contenu a été mis à jour le 9 décembre 2015 à 16 h 59 min.


text:
Port Talbot's Enterprise Zone will Encourage Economic Growth and Employment
Beth Perry , 21st March 2016
The Leader of Neath Port Talbot Council has welcomed announcements by the Welsh and UK Governments to boost the local and regional economy
The establishment of a new Enterprise Zone at Port Talbot Waterfront, announced by Edwina Hart, Welsh Government Minister for Economy, Science and Transport means the area will benefit from a range of incentives to stimulate economic growth and develop new employment opportunities.
The Port Talbot Waterfront Enterprise Zone will encompass established employment sites such as Baglan Energy Park, Baglan Industrial Estate and Port Talbot Habourside as well as potential development sites opened up by developments such as Harbour Way.
Work to establish the enterprise zone has been undertaken through close working between Neath Port Talbot Council and the Welsh Government and through the Business and Supply Chain work stream of the Tata Steel Task Force.
Council Leader Cllr Ali Thomas OBE said:
"We are working closely alongside partners to ensure Port Talbot, the County Borough and the wider region can meet the challenges posed by continuing uncertainty in the global economy."
"The establishment of an Enterprise Zone for Port Talbot will ensure that the area continues to diversify its economy and encourages further investment and the creation of high quality employment.
"Past and ongoing infrastructure projects such as Harbour Way, the new Baglan Energy Park Bridge, Harbour Walk, the Vibrant and Viable Places programme and the redevelopment of Port Talbot Parkway mean that the Enterprise Zone and the town is an increasingly well connected location for business to flourish."
Cllr. Thomas also welcomed the UK Government budget announcement which saw Chancellor of the Exchequer George Osborne give the go-ahead for talks to begin on the ambitious 'Internet Coast' City Deal proposed by the Swansea Bay City Region Board.
"I am delighted we can now enter formal discussions on what is a very exciting and forward thinking proposal to harness digital infrastructure and technologies on an unprecedented scale."
Swansea Accountancy Practice Offers Essential Guidance in Wake of Charity Commission Alert
4th May – Caerphilly
Business Networking Morning
From a seaside town in South Wales, Beth is a first language Welsh speaker with a passion for writing and a savvy working of social media.
After graduating from Cardiff University in 2017 with a degree in English Literature, Beth is continuing her studies with a master's degree in International PR and Global Communications Management at Cardiff. During this time, Beth has held several part-time jobs in the retail and sales sectors, as well as a summer teaching in the USA which has given her a wealth of skills and experience.
Beth has a keen sense of what makes a good story and enjoys consuming news in all formats, whether it be through social feeds or a good old fashioned newspaper. To that end, she is full of quirky facts and snippets of knowledge that would serve just as well in a pub quiz as it would a compelling piece of content; she enjoys watching quiz shows like Only Connect and University Challenge to test herself.
In her spare time Beth enjoys cooking and baking, especially when it comes to trying new recipes… as long as she doesn't have to clean up the mess afterwards!
£1.6m Investment Set to Create 100 New Jobs Boost on Haven Waterway Enterprise Zone
City Region Plan for Cardiff, Newport and Bristol Unveiled
Newport's Celtic Manor Convention Centre Gets Green Light


text:
/**
 * Message Dumper
 * Helps with debugging by logging every single Steam message.
 */
exports.name = 'punk-message-dumper';

exports.plugin = function(API) {
  const log = API.getLogger();
  const utils = API.getUtils();
  const Steam = API.getSteam();

  const ignored = {};
  ignored[Steam.EMsg.Multi] = true;

  API.registerHandler({
    emitter: 'client',
    event: 'message'
  }, (header) => {
    if(header.msg in ignored) {
      return;
    }

    log.debug('Received message %d (%s).', header.msg, utils.enumToString(header.msg, Steam.EMsg));
  });
};

redpajama_set_name: RedPajamaGithub


Pure, potent, intensive single dose capsules. This innovative CLX Complex delivers the restorative power of Ceramides and essential lipids to help strengthen skin's barrier against the visible signs of time.- Visibly improves skin tone and clarity.- Supports skin's own natural collagen levels for a firmer, more resilient look.- Works to minimise the appearance of fine lines and wrinkles.- Retexturises skin for a noticeably softer, smoother look. Fragrance, preservative and emulsifier free.
To use: Apply daily to cleansed skin. To open capsule, twist tab around twice to snap off. Smooth serum over cleansed face and neck. Follow with your moisturiser.
To use: Apply daily to your cleansed skin before sun exposure and after using Ceramide Capsules.
A night time moisture treatment for face and neck to instantly infuse skin with ceramides for a youth restoring boost. Helps skin recover overnight as it continuously replenishes skin's moisture equilibrium and supports skin's own natural protective moisture barrier all night long. Wake up to skin that looks firmer and more lifted, rested and revitalised.
To use: Apply evenings. Smooth onto cleansed skin after applying ceramide capsules.


Today's Petrol price in Yavatmal (Maharashtra) is Rs. 79.47 per Litre. Last change in Yavatmal petrol price was on April 19, 2019 and it was increased by +0 rupees. In addition, we bring you the most recent petrol price changes in Yavatmal city. The petrol price is inclusive of Maharashtra state taxes.


It's been a couple months since I last posted something on my blog. I said I would resume on December 1, so this post is a couple days early.
I've learned a ton of things. I tried all sorts of interesting changes to my planning and my routine. Some worked well, some didn't, but it was still a great experience.
I learned how to better manage my time and how to get things done. I can now create working plans both for short-term and long-term goals. I have figured out how to make more time in my day for all the things I want to do.
The most important thing I have learned is this: no matter how good your planning is, if your priorities aren't right, you won't get much done.
I worked really hard so squeeze as many useful minutes out of my day as possible. I've perfected my schedule to be as efficient as it can be. And yet I still did not have enough time for all I wanted to do.
My problem was trying to do too many things. The real secret to making time is not so much getting rid of time-wasting habits. It is all about picking the one thing to spend your time on. It could be two or three things, but the more you add, the less time you have for each.
I love writing this blog, and it is something that I want to continue doing in the long term. However, there are other things in my life that are more important at this time.
I could continue my balancing act and try to do the impossible. Or I could honestly admit that once I list my priorities out, writing ends up just below the cut-off.
It is deeply disappointing to realize and admit that I cannot do everything. I wish I could, but all the evidence is pointing to the fact that I simply need to focus on fewer things, and change my focus from time to time.
One day, I'll come back and write more. I still have a lot to say. I don't know when that day would be, but it will come when the time is just right.
I encourage you to think about your time and your priorities. Would you change anything about them to become happier?


text:
ACCEPTED

#### According to
Index Fungorum

#### Published in
null

#### Original name
Asterina cissi S. Hughes

### Remarks
null


text:
Национален отбор по волейбол на Холандия може да се отнася към:
 Мъжки национален отбор по волейбол на Нидерландия
 Женски национален отбор по волейбол на Нидерландия


text:
Kleptopharmacophagy is a term used for describing the ecological relationship between two different organisms, where the first is stealing the second's chemical compounds and consuming them. This scientific term was proposed by Australian, Singaporean, and American biologists in September 2021 in an article that was published in the journal Ecology by the Ecological Society of America. The phenomenon was first noticed in milkweed butterflies that were attacking caterpillars and drinking their internal liquid, proposedly to obtain toxic alkaloids used for defense, as well as for mating purposes.

Characteristics
Kleptopharmacophagy is a generic term and a scientific neologism, that is used to describe the phenomenon of chemical theft between living organisms. This special type of behavior is something new for researchers, as it does not match the traditional descriptions of biological interaction. Kleptopharmacophagy cannot be defined simply with a use of classical ecological relationships and their descriptions, such as predation, parasitism and mutualism. Besides being an interspecific interaction, kleptopharmacophagy can also be seen as a cannibalizing act (hence an intraspecific act), with adults attacking larvae of their own species.

In butterflies

Kleptopharmacophagy has only been reported for butterflies in the subfamily Danainae, more commonly known as the milkweed butterflies. The phenomenon was first described in butterflies that were observed scratching and feeding on live larvae of other milkweed butterflies. The initial discovery was made in year 2019 in forests of North Sulawesi, in Indonesia, when two researchers noticed that milkweed butterflies of different species, well-known for their toxicity and bright warning colours (aposematism), were interacting with the larval stages of other butterflies. The adult insects were attacking and harassing the caterpillars by scratching at them with tarsal claws on their legs. The butterflies then imbibed the juices from the wounded caterpillars with their long and curved proboscis. Caterpillars that were targeted appeared to range from living to dead and dying individuals. Live caterpillars were observed to contort their bodies in an attempt to deter scratching, but usually succumbed to the repeat harassment.

There is also one recorded example of kleptopharmachophagy occurring between two adult butterflies; a male Ideopsis vitrea vitrea was feeding on a liquid, oozing from the wings of the male I. blanchardii blanchardii.

Role

It is thought that such chemical theft serves at least two roles. By feeding on toxic caterpillars, the adult butterflies acquire additional toxins, which serve as a protection against predation. The stolen toxins, mostly pyrrolizidine alkaloids (PAs), are also of great importance for male butterflies, which use those compounds for producing mating pheromones. Additionally such chemicals are components of so-called nuptial gifts, consisting of male sperm and some nutrients, that are given from males to females during mating. Significance of the alkaloid use for male butterflies can be demonstrated through observation of male danaine butterflies congregating in large numbers at alkaloid producing plants, where they spend hours fervently scratching and liberating plant juices.

Similar behaviour
Milkweed butterflies have also been previously seen feasting on some moribund pyrgormorphid grasshoppers, that are known to contain toxic alkaloids, as well as obtaining desired chemicals from carcasses of dead insects of different taxa. Note that in this case, the term necropharmacophagy may be more appropriate, since butterflies feed on the dead animals.

Similar behavior was observed with adult butterflies scratching leaves of different plants that possess various toxins in their vegetative organs (so-called leaf-scratching). Adult male danaine butterflies use their sharp tarsal claws to scratch and damage leaves of plants that contain high levels of pyrrolizidine alkaloids, thereby liberating the juices for consumption. Since kleptopharmacophagy was recorded and described only a few times, it is thought that obtaining such chemicals by feeding on plants with alkaloids is a common and more frequent way of stocking toxins. Kleptopharmacophagy is proposed to be an alternative way of acquiring these chemicals.

References

Bibliography

Animal cannibalism
Eating behaviors
Butterflies
2021 in biology


Bob's Red Mill has been, and continues to be one of SpectraGraphics's most valued and long term clients. Despite multiple offers founder Bob Moore recently gave his $100 million company to his employees to continue on with his legacy. Bob is much more than a client to many of us here at SGI, he is a friend, an inspiration and an ideal image of what American businessmen can and should be.
In an era of corporate greed and global recession Bob Moore is a shining example of what's right about America. He has recently released his autobiography "People Before Profit – The Inspiring Story of the Founder of Bob's Red Mill". We strongly encourage anyone managing a company to read it, and live it. It's available on Amazon in both electronic format and hard copy, the link is below.
Our sincerest thanks and best wishes to Bob and his wife Charlee, and to all the employees of Bob's Red Mill for a bright future.
We are very pleased to announce that SpectraGraphics has added a new state of the art Mark Andy P5 series flexographic printing press. The new unit combines cutting edge drying technology with quick change over and auto registration print stations. Incredibly fast speeds are now achievable for long runs, combined with fast copy changes, enhanced quality and reduced scrap rates for overall cost reduction.
This equipment has an impact in other areas as well. The increased capacity in both web width and speed results in reduced lead times and lowered set up costs on other equipment, such as that utilized for expanded content products. We are now branching in to other printed market segments such as unsupported films for continuous labeling and pouches.
Please contact us for more details, we look forward to helping you grow your business!
The SpectraGraphics family was very proud to be 2011 sponsors for a Shawnee Mission Head Start Family. The program's mission is "to provide quality early childhood and comprehensive child and family services, which empower low-income families and their children to maximize their success." Eligible families have an annual household income of under $18,000 despite full time employment, so our gifts of clothing, toys, food and everyday products were greatly appreciated. It's easy to over look the fact that some families live without the means to cover basic human needs, especially over the holiday season.
SpectraGraphics Inc. is proud to once again be a sponsor of the Children's Mercy Hospitals "Red Hot Nights" event. The annual fundraising event surpassed it's goal of $1,000,000 this year benefiting the new east facility. Children's Mercy hospitals and clinics provide some of the best care in the nation to children and their families in need, and the new facility brings this care closer to home for many families in eastern Jackson County MO.
We are very pleased with this year's turnout and contributions, our sincerest thanks to those clients that attended the event and donated to such a great cause!


Foulden, Scottish Borders, TD15 1UG.
Tweedmouth, Scottish Borders, TD15 2AN.
Foulden Newton, Berwick-upon-Tweed, Scottish Borders, TD15 1UL.
Berwick-upon-Tweed, Scottish Borders, TD15 1ES.
Norham, Scottish Borders, TD15 2LD.


text:
namespace Errors {
	const error_data
		EntityNotFound = { 404, "Entity does not exist in this system!" },
		EntityMissingInit          = { 301, "Entity behavior component does not have an appropriate init function" },
		EntityInitException        = { 302, "Entity behavior component init() threw an exception" },
		EntityInitUnknownFailure   = { 309, "Entity behavior component init() failed in an unexpected way" },
		EntityUpdateException      = { 312, "Entity behavior component update() threw an exception" },
		EntityUpdateUnknownFailure = { 319, "Entity behavior component update() failed in an unexpected way" };
}

typedef uint32_t EntityId;
class EntitySystem;
struct ControllerInstance;

// Instances of entities
struct Entity {
// === Metadata ===
	const EntityId id;
	EntitySystem* system;

// === Physics Data ===
	Point2 position       = { 0.f,0.f };

	Vector2 velocity      = { 0.f,0.f };
	Vector2 acceleration  = { 0.f,0.f };

	// Needed for tunnelling prevention and effects such as motion blur.
	Point2 last_pos       = { 0.f,0.f };

	// Other movement stuffs
	AABB vel_range = { -INFINITY, INFINITY, -INFINITY, INFINITY }; // Velocity range

// === Transform Data ===
	float rotation = 0.f;
	Vector2 scale = { 1.f, 1.f };

// === Render Data ===
	const Sprite* sprite = nullptr;
	const Animation* animation = nullptr;
	const Frame* frame = nullptr;
	uint32_t anim_frame = 0;
	float frame_time = 0.f;

	int z_order = 0;

// === ColliderChannel flags ===
	uint64_t channel_mask = 0xFFFFffffFFFFffff;
	uint8_t channel_id;

// === Enable/Disable flags ===
	bool physics_enabled = true;
	bool collision_enabled = true;
	bool animation_enabled = true;
	bool rendering_enabled = true;
	bool solid = true;

// === Script Interface ===
	asIScriptObject* rootcomp = nullptr;
	asITypeInfo* rootclass = nullptr;
	asIScriptFunction* updatefunc = nullptr;

// === Functionality ===
	Entity(EntityId id, asIScriptObject* behavior);
	~Entity();

	// In order to keep errors as return values (not throwing exceptions), init must be separate;
	Result<> init(asIScriptEngine* engine);
	Result<> update(asIScriptEngine* engine, float delta_time);

	void render(GPU_Target* screen) const;

	inline Transform get_transform() const {
		return Transform::scal_rot_trans(scale, rotation, position);
	}
};

struct LevelInstance;

class EntitySystem {
	typedef std::vector<Entity*> EntityList;
	typedef EntityList::iterator EIter;

private:
	BucketAllocator<Entity> allocator;
	EntityList entities;
	EntityId next_id;

public:
	bool ordered = false;

	EntitySystem();
	EntitySystem(const EntitySystem&) = delete;
	EntitySystem(EntitySystem&&) = default;

	~EntitySystem();

	Result<Entity*> spawn(asIScriptObject* rootcomp);
	Result<> destroy(EntityId id);
	Result<> destroy(Entity* ent);

	void update(asIScriptEngine* engine, LevelInstance* level, const float delta_time);

	/// Iterator set for entities - allows for interleaved rendering
	std::pair<EIter, EIter> render_iter();
};

void RegisterEntityTypes(asIScriptEngine* engine);

// TODO: SOA/SIMD particle system


Sterling Imageworks Photography is owned by Callie and Jaison Sterling, a husband + wife team out of North Little Rock, Arkansas. Their goal is to help you tell your wedding story, the way you want it told. They specialize in wedding & lifestyle photography & offer a variety of packages to fit your photography needs. With experience in wedding photography since 2010, the pair has over 150 weddings under their belt. Contact Sterling Imageworks by e-mail at jc@sterlingimageworks.com or by phone at 501-413-0841.


Sorry, we are currently sold out of 'Outwit Outlast Outsmart the Test - Sharpened'. Please check back later.
These Outwit Outlast Outsmart the Test motivational pencils are great for classroom incentives and school supplies. Pencils are made from safe, non-toxic paint and materials. #2HB lead.


2018 sees the 150th anniversary at the All England Lawn Tennis & Croquet Club- now more famous for the Wimbledon Tennis Championship. Any croquet player of course is aware that the club started out as a Croquet Club before evolving into the Tennis Championship. The club is having a exhibition of its 150 years at the museum, including a section on croquet titled 'Through the Hoops: croquet at Wimbledon'. The exhibition runs from the 22nd of March 2018 to the 3rd of February 2019.
Here at the shop we list some historic prints including a interesting one of croquet at the All England Club. Originally published in 1870 by The London Illustrated News this print is at £6.00 including delivery.
We are looking out on lawns covered by snow and next week the Cheltenham Club lawns are closed for maintenance. So, unfortunately, although the shop is open customers will not be able to try out our mallets on the lawns.
Lawns will be open, dependent on weather from the 12th March.
The Croquet Association is pleased to announce the appointment of Mark Suter as the CA Manager who will take up his appointment on 26th February 2018. He succeeds Liz Larsson who is leaving us after almost 10 years of loyal and dedicated service.
He has spent 25 years abroad as a Business and Marketing Consultant in the Finance sector working from Cape Town and Dublin for 10 and 15 years respectively.
Mark has enjoyed a long involvement with Croquet and is a top ranked player of both Association and Golf Croquet. As a teenage member of Bristol Croquet Club in the nineteen-eighties he was among the top ranks of Association Croquet players. Since his return to England he began his comeback to the tournament circuit in early 2017 as a member of the Nailsea and Cheltenham clubs. His 2017 season culminated with winning the Chairman's Salver.
We would like to wish a very happy new year for our customers at the Croquet Association. We are well aware that a bit of cold weather (frost permitting!) does not stop a hardy croquet player in the winter months. At the shop we can offer a wide range of fleeces, pullover jumpers and reversible gillets to help keep you warm through the winter. These can be found in the Croquet clothing section of the shop.
Please note that the shop will be closed on Christmas Day and Boxing Day Bank Holidays, opening again on Wednesday the 27th of December.
Although the shop will be open to take orders and queries, any deliveries would not be sent until after the New Year on Tuesday the 2nd of January 2018.
We would like to wish all our customers a merry Christmas and a happy New Year.
The seasonal rush is well under way and as usual deliveries are subject to delays but we will do everything we can to ensure you orders are delivered on time.
20th December for UK postcodes.


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