text:
Bath City Apartments (Argyle Street)
|Prices will be charged in United Kingdom Pounds
|Full Rate
|Wed 15 Aug 2018
|Thu 16 Aug 2018
|Fri 17 Aug 2018
|Sat 18 Aug 2018
|Sun 19 Aug 2018
|Mon 20 Aug 2018
|Tue 21 Aug 2018
|Wed 22 Aug 2018
|Thu 23 Aug 2018
|Fri 24 Aug 2018
|Sat 25 Aug 2018
|Sun 26 Aug 2018
|Mon 27 Aug 2018
|Tue 28 Aug 2018
Double Room + Sofa bed (AS) standard rateDetails
|Move the mouse over the price for inclusions, occupancy and minimum stay
Property Information
Bath City Breaks (Argyle Street)
Accommodation Details
Terms & Conditions
Booking Terms & Conditions
Our booking terms and conditions are listed below. Please read these carefully as they form the basis of the contract between us.
• All bookings are subject to final confirmation by us.
• The period of rental shall be from 3.00pm on the day of arrival until 11.00 am on the day of departure.
• All prices are in Pounds GB and are fully inclusive of electricity, full central heating, bed linen and towels. We provide one bath sheet and one hand towel per person.
• Upon receipt of your booking we will email to confirm you selection. We will provide you with a total bill. For your booking to be guaranteed we require an immediate 30% deposit and the remaining full balance to be paid before 6 weeks of your arrival. If your booking is within 6 weeks we require the total bill to be paid in full in order to guarantee your booking. Your booking will not be finally confirmed until we receive your payment.
• All payments shall be made by cheque payable to Mr A. I. F. Forsyth'. Or Bank Transfer to Barclays – Mr A. I. F. Forsyth – A/C 43636658 – S/C – 20-05-06 (Remember to include your name in the Reference). All bank charges must be paid by the client.
• In the unhappy event of a can¬cel¬la¬tion we will only refund any monies paid if we are able to re-let the prop¬erty (less a £30 can¬cel¬la¬tion fee)
• The hirer must personally stay at the accommodation throughout the holiday and is solely responsible for the whole party. Assignees and sub-tenants are prohibited. No more than the stated number may stay at the property.
• Please familiarize yourself and the other guests with the procedures in the unlikely event of a fire. These are provided in the welcome pack and should be noted.
• A highchair is available at no extra charge, but please bring your own cot with its own linen.
• Children of all ages are welcome. But please be careful of the low windows and access to the balcony and make sure children are properly supervised to prevent accidents.
• Re accessibility, the apartment is not suitable for wheelchair users, but may be suitable for persons with lesser disabilities. If you have someone less able in your party, please contact us to discuss your requirements before making a booking.
• Bookings will not be accepted from groups of single people under the age of 21 years.
• For insurance reasons, only the number of guests specified at the time of booking may stay, unless you properly notify us of changes well in advance.
• For the comfort of all of our guests the apartment is strictly non-smoking.
• Registered assistance dogs apart, we do not allow pets.
• The hirer undertakes to take all reasonable care in the use of the property and its contents, including the security of the property. The property should be left in the condition it was found, any excessive cleaning will be charged to the client. The hirer will report any damages, losses or breakages as soon as they occur. The Company reserves the right to charge for repairs, losses or replacements necessitated by the negligent act or omission of any of the hirer's party, guests or invitees. If necessary, this may include an administration charge.
I confirm that I have read and accept the booking conditions.


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A new survey from E-Poll concludes, not surprisingly, that on-air promotion and word of mouth are the major ways people find out about new shows. Reviews from critics were far less important, and even less important than that? Blog posts! I don't take any personal offense to that because a.) it seems very likely true and b.) we don't typically spend any time promoting new shows, instead just waiting until they air to comment on the ratings.
But here's what they had to say about Internet promotions:
Current Internet promotions are largely ineffective, as the Internet falls far behind television as a source of new program awareness, at about 15% recall vs. 94% for TV ads. Online advertisers have an opportunity to better engage potential viewers by discarding re-purposed 30-second spots and creating custom content that lives in the unique online environment.
Here's the full list:
(METHODOLOGY: This online survey was conducted among a nationwide, representative sample of 1644 individuals age 13 – 54. The margin of sampling error at 95% confidence for aggregate results is +/- 3%. Sampling error is larger for subgroups of the data. The survey was fielded May 21 – 26, 2009.)
Someday DVR and VOD will be a bigger promotional vehicle for new shows
Although outside of the people who write press releases, the TV industry is generally fearful of the impact of DVR and other technology, the E-Poll survey concludes that when it comes to watching new shows, people who use DVRs and Video On-Demand are much more likely to check out new shows. Nearly twice as likely as non-users, according to the survey. E-Poll views that as a major opportunity when it comes to marketing of new shows. While I generally agree, right now it seems there are too few DVR users (and far fewer VOD users) to make much of a dent, particularly when it comes to broadcast shows. But we have seen cable networks over the last couple of years premiere shows first on VOD ( Showtime and Nurse Jackie, for example)

subset: commoncrawl


text:
Item# 0607845013068
€ 55,00
Product Actions
Additional Information
NARS Candles were created to elevate the senses and instill a peaceful ambiance. Each possesses a uniquely delicious aromatic scent formulated to enhance the mood, inspire the senses and provoke individual memories. Encased in white, black or red matte glass with the NARS logo, each paraffin-free candle is composed of rich essential oils, allowing for an optimum burn time of 70 hours. Top notes of a teasingly sweet blend of citrus are complimented with hints of pomegranate and balanced by rich, exotic floral notes of rose, jasmine and geranium. Full-bodied base notes of amber and vanilla add warmth to the tantalizing scent for an overall sweet and seductive sensory experience.
Fragrance makes us dream -- François Nars


text:
.ee has a new registrar - Web Commerce Communications Ltd.
We signed a .ee accredited registrar contract with Web Commerce Communications Ltd. (WEBCC).
WEBCC uses a global identity WebNIC and is an Internet service provider focusing on domain name wholesale service and digital brand management.
Established in 2000, WEBCC has offices in Singapore, Taiwan and Malaysia. WEBCC is an ICANN accredited registrar.
WEBCC manages over 700,000 domain names, over 3,000 resellers and over 60 countries.


text: Mass for James F. Ward Sr., 80, a retired Chicago Fire Department lieutenant, will be said at 10 a.m. Tuesday in St. Anne Catholic Church, 168th Street and Dixie Highway, Hazel Crest. Mr. Ward died Friday in the University of Chicago Hospitals & Clinics. After 28 years with the Chicago Fire Department, he retired and became a foreman for Peoples Gas, Light & Coke Co., a job from which he later retired. Mr. Ward is survived by his wife, Marcella; 5 sons, James F. Jr., Bernard, Thomas, John and Neil; a daughter, Susanne Brown; 25 grandchildren; 2 great-grandchildren; 3 brothers; and 4 sisters.


text:
Patent application title: Viral Immunogenic Compositions
Henry J. Smith (Temecula, CA, US)
Henry J. Smith (Temecula, CA, US)
James Roger Smith (Temecula, CA, US)
IPC8 Class: AA61K39145FI
USPC Class:
Class name: Drug, bio-affecting and body treating compositions preparations characterized by special physical form liposomes
Publication date: 2015-11-19
Patent application number: 20150328305
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Disclosed herein are immunogenic compositions for producing immediate and
sustained immunity to infectious viral and bacteriological pathogens. A
univalent immunogenic composition is disclosed comprising an isolated
antigen and a polynucleotide formulated into a nanoparticle or liposome.
Furthermore, multivalent immunogenic compositions are disclosed
comprising multiple univalent immunogenic compositions. Also disclosed,
are methods of inducing protective or therapeutic immune responses in
individuals comprising administering one or more univalent immunogenic
1. A method of inducing a protective or therapeutic immune response to
influenza in an individual in need thereof comprising: administering one
or more univalent immunogenic compositions to the individual, wherein a
univalent immunogenic composition comprises (i) an isolated influenza
virus antigen, and (ii) a polynucleotide; each bound to the surface of,
or contained within, a nanoparticle, or each contained within a liposome;
thereby inducing a protective or therapeutic immune response to influenza
in the individual.
2. The method according to claim 1, wherein a first univalent immunogenic composition comprises a first antigen.
3. The method according to claim 1, wherein a second univalent immunogenic composition comprises a second antigen.
4. The method according to claim 1, wherein the one or more univalent immunogenic compositions further comprise an adjuvant.
5. The method according to claim 4, wherein the adjuvant is alum.
6. The method according to claim 4, wherein the adjuvant is bound to the surface of, or contained within the nanoparticle, contained within the liposome, or admixed with the nanoparticle or liposome.
7. The method according to claim 1, wherein the influenza virus antigen is a hemagglutinin, a neuraminidase, or a matrix protein.
8. The method according to claim 1, wherein the polynucleotide is a synthetic polynucleotide comprising double stranded DNA or double stranded RNA.
9. The method according to claim 8, wherein the double stranded DNA or double stranded RNA comprises nucleotide repeats.
10. The method according the claim 8, wherein the polynucleotide is polyinosinic-polycytidylic acid (poly-IC) or polyinosinic-polycytidylic acid-poly-L-lysine (poly-ICLC).
11. The method according to claim 1, wherein the liposome is a self-assembling liposome system.
12. The method according to claim 1, wherein the liposome further comprises monophosphoryl lipid A or pullulans.
13. The method according to claim 1, wherein the one or more univalent immunogenic compositions are administered by a method selected from the group consisting of scarification, intradermal injection, intramuscular injection, sub-lingual administration, and inhalation.
14. The method according to claim 1, wherein the one or more univalent immunogenic compositions are administered by injection to the individual at separate sites on the individual.
15. The method according to claim 1, wherein the one or more univalent immunogenic compositions are administered at different times.
16. The method according to claim 1, wherein the one or more univalent immunogenic compositions are administered substantially simultaneously.
17. The method according to claim 1, wherein the one or more univalent immunogenic compositions are administered by injection to the individual at the same site on the individual.
18. The method according to claim 1, wherein the one or more univalent immunogenic compositions are combined prior to injection into a multivalent immunogenic composition.
19. The method according to claim 1, wherein the method comprises administering two or more univalent immunogenic compositions, wherein each of the univalent immunogenic compositions comprises a different influenza virus antigen.
[0001] This application is a divisional of U.S. patent application Ser. No. 13/566,663 filed Aug. 3, 2012, which claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application 61/514,613 filed Aug. 3, 2011, the entire contents of both of which are incorporated by reference herein.
[0002] The present application is drawn to immunogenic compositions for preventing and treating Influenza virus infection, and methods of making and using the immunogenic compositions.
[0003] Every year many people catch "the flu" and for most it is a self-limiting illness with recovery occurring between two to three weeks. Influenza is however, a potentially more severe infectious disease which is highly contagious. It is estimated that there are between three to five million cases of severe flu worldwide resulting in up to 500,000 deaths annually. There are two types of influenza viruses that cause epidemic human disease: the Influenza A virus which has many different strains, and the Influenza B virus which consists of a single strain. Current flu vaccines are a mixture of viral antigens prepared from selected strains of influenza A virus and from the single strain of the influenza B virus. The current vaccination programs are aimed at containing the seasonal flu. Health authorities from many countries collaborate with one another to predict the strain of influenza A virus that is likely to cause flu in the upcoming months. For the most part, these types of vaccination programs have worked well to contain the seasonal flu, although they become less effective when the flu epidemic is caused by a different influenza A strain than was predicted. This is because vaccination against one influenza A strain affords little if any immunity against a different influenza A strain. Once the strain is identified it takes about six months to manufacture large quantities of the appropriate vaccine. As such, one of the drawbacks of these vaccination programs is the time it takes to formulate a vaccine which may not even provide any protection against the influenza A strain causing seasonal flu in a particular season. For instance, if it takes six months to formulate a vaccine which provides little or no immunity because the predicted strain is not the one causing the infection, it will take another six months to develop the appropriate vaccine at which point there may be a threat for a major flu pandemic.
[0004] It would therefore be desirable to develop an effective immunogenic composition that is tailored to any flu strain that may arise and that can be produced in large quantities and deployed rapidly. Further, it would be desirable if the immunogenic composition could elicit an immediate and prolonged immunity to the flu virus.
[0005] The desirability of developing such an immunogenic composition becomes even more urgent when one considers that every ten to twenty years a flu strain arises that infects a large proportion of the world's population and has the potential to kill tens of millions of people. For example, within the last century there have been three major flu pandemics: the Spanish Flu in 1918-1920 which killed 40 million to 100 million people; the Asian Flu in 1957-1958 which killed 1 million to 1.5 million people; and the Hong Kong Flu in 1968-1969 which killed about 1 million people.
[0006] Another major obstacle in developing an effective vaccination program is that the influenza A virus is constantly changing. For example, it is known that the influenza A virus may undergo successive point mutations during replication (i.e. genetic drift) and can gradually acquire the capacity over time to become more virulent. Another way in which new strains of flu virus appear is when different flu strains may co-infect a host and exchange genetic information with each other (i.e. genetic shift) resulting in the appearance of a new flu strain with different infective properties and pathogenic potential from either of its parent strains.
[0007] Different strains of the influenza A virus are known to infect a variety of animal species, including birds and pigs, and some of these strains may cross specie lines and infect humans. If the infected human also happens to be co-infected with another strain of influenza virus there is a possibility that the two viral strains may exchange genetic information resulting in the appearance of a new strain. For example, there is great concern that the avian flu virus found in domestic and wild birds may infect a human who is carrying a human flu virus strain and exchange genetic information to form a new flu viral strain with the capacity to propagate from human to human resulting in a worldwide pandemic.
[0008] There are intensive efforts being made to develop antiviral drugs and vaccines to combat the threat of a worldwide pandemic. Much of the research on flu vaccine is based on traditional methods of growing the virus in embryonated eggs or in tissue cultured cells and then extracting the viral proteins to prepare a vaccine. This has worked fairly well for seasonal flu vaccines where health agencies are able to identify new strains of flu virus as they arise and have sufficient time to prepare the appropriate vaccine. However, the vaccination program still has to be repeated each year as immunity to one strain of the flu virus will not fully protect against any new strain that develops.
[0009] Another major obstacle with the traditional vaccination program is that because of mass transit and the large numbers of air travelers, any flu strain that arises in one country can quickly spread among other countries and infect the general population. Moreover, because of the many potential permutations of influenza virus strains that can arise, it is difficult to prepare a vaccine in a timely fashion using the traditional method of growing the virus in embryonated eggs or culturing viral infected cells. Thus, the time frame for identifying new viral strains and preparing large amounts of the correct vaccine has grown increasingly short which continues to be one of the major hindrances facing the traditional vaccination program. There are also very stringent manufacturing safety protocols that must be employed throughout production when a pathogenic virus is used to prepare a vaccine. Even the more recent methods of using genetic engineering to prepare recombinant viral antigens still require several months to prepare a suitable vaccine that may not be deployed in time to be effective.
[0010] The current vaccination programs that were developed for the seasonal flu are unable to produce the large quantities of vaccine in the limited time available to protect against a flu pandemic. Even the most current vaccine production methods using cell culture requires about 4 months to produce the appropriate vaccine (Novartis 2008). This time frame is clearly inadequate when the response time required to prevent a flu pandemic is measured in days.
[0011] The difficulty is further compounded because the current vaccines are only designed to elicit a classical immune response which can take several weeks to develop after vaccination. Moreover, to develop full immunity two doses of vaccine are often required. For example, The World Health Organization reported in 2007 that "It is unlikely that a potential pandemic could be successfully contained through the use of vaccination alone. Vaccination, with some exceptions, is not normally used to "contain" outbreaks of seasonal influenza. Full immunity is likely to require two doses of vaccine, and to take 3 weeks to develop after the first vaccination. Moreover, the degree of antigenic match between stockpiled vaccine and a potential H5N1 influenza pandemic virus cannot be known. There will also be considerable logistical challenges in vaccinating large numbers (possibly millions) of people over a relatively short period of time."
[0012] In view of the deficiencies of the current vaccination programs it would be desirable to develop an immunogenic composition that could provide immediate and long-term immunity to any flu strain that should arise. It would also be desirable to be able to produce large quantities of the appropriate flu immunogenic composition very quickly so that it can be deployed in time to be effective.
[0013] This application discloses a method of preparing a multivalent immunogenic composition against the influenza A virus.
[0014] Disclosed herein, is a univalent immunogenic composition comprising an isolated antigen and a polynucleotide formulated into a nanoparticle or liposome.
[0015] In one embodiment, the univalent immunogenic composition further comprises an adjuvant. In another embodiment, the adjuvant is alum. In another embodiment, the adjuvant is formulated into the nanoparticle or liposome or admixed with the nanoparticle or liposome formulated univalent immunogenic composition.
[0016] In another embodiment, the isolated antigen is an influenza virus antigen such as a hemagglutinin, a neuraminidase, or a matrix protein.
[0017] In one embodiment, the polynucleotide is a synthetic polynucleotide comprising double stranded DNA or double stranded RNA. In another embodiment, the double stranded DNA or double stranded RNA comprises nucleotide repeats. In yet another embodiment, the polynucleotide is poly-IC or poly-ICLC.
[0018] In another embodiment the liposome is a self-assembling liposome system. In yet another embodiment, the liposome further comprises monophosphoryl lipid A or pullulans.
[0019] Also disclosed herein, is a method of inducing a protective or therapeutic immune response in an individual comprising administering one or more univalent immunogenic compositions, wherein a first univalent immunogenic composition comprises a first antigen. In another embodiment, a second univalent immunogenic composition comprises a second antigen. In another embodiment, a third univalent immunogenic composition comprises a third antigen.
[0020] In another embodiment, the one or more univalent immunogenic compositions are administered by a method selected from the group consisting of scarification, intradermal injection, intramuscular injection, sub-lingual administration, and inhalation. In other embodiments, the one or more univalent immunogenic compositions are administered at different times or substantially simultaneously. In another embodiment, the one or more univalent immunogenic compositions are each administered by injection to the individual at the same site on the individual or at different sites on the individual. In another embodiment, the one or more univalent immunogenic compositions are combined prior to injection into a multivalent immunogenic composition.
[0021] Also disclosed herein, is a multivalent immunogenic composition comprising two or more univalent immunogenic compositions, wherein each of the univalent immunogenic compositions comprises a different antigen.
[0022] In yet another embodiment, the approach of stockpiling all the components required to prepare a complete univalent or multivalent immunogenic composition or the univalent immunogenic compositions themselves is disclosed. The components are viral antigen and polynucleotide packaged into a nanoparticle or liposome. Once an epidemic flu virus is identified, the stockpiled immunogenic composition components, or univalent immunogenic compositions, can be quickly assembled into the appropriate univalent immunogenic compositions directed against the identified viral antigens. The selected univalent immunogenic compositions are then administered separately or they may be combined to form a multivalent immunogenic composition before administration.
[0023] The same general principles enumerated herein can be applied to preparing immunogenic compositions against other viral and bacterial pathogens. It can be particularly advantageous when applied to vaccination programs against potential pandemics and the threat of bioterrorism.
[0024] Disclosed herein is a comprehensive integrated approach to developing an effective vaccination program that can be employed to protect against influenza. The same general principles can also be employed to prepare immunogenic compositions against other viral and microbiological pathogens. As used herein, an "immunogenic composition" refers to a composition capable of inducing a protective or therapeutic immune response, with or without an adjuvant, and includes, in part, an antigen against which the protective or therapeutic immune response is elicited. An immunogenic composition is an example of an immunogenic composition.
[0025] First, a number of basic univalent immunogenic compositions are prepared with each immunogenic composition directed against a single antigen selected from the group of hemagglutinin (H), neraminidase (N), and matrix (M) antigens of the influenza A virus. An individual's immune response is enhanced by presenting only one purified antigen at a time to an antigen processing and/or immunocompetent cell. Rapid and persistent immunity is achieved by incorporating a double-stranded polynucleotide chain with each specific viral antigen. The combined antigen and polynucleotide moieties are incorporated into a nanoparticle or liposomal univalent immunogenic composition formulation. Administration of the immunogenic composition stimulates an immediate protective interferon response followed by the classical immune response to the antigen. To shorten the response time to the threat of an epidemic, the individual components of the univalent immunogenic composition i.e. viral antigen, polynucleotide, and nanoparticle or liposome supplies are separately stockpiled and stored. Once the suspect flu virus strain is identified the appropriate immunogenic composition components are then assembled into separate univalent immunogenic compositions. Individuals may receive one or more univalent immunogenic compositions directed against different flu antigens to obtain broad immunity. Several different univalent immunogenic compositions may also be combined to produce a multivalent immunogenic composition for convenience in administration. Vaccination may be by dermal, sub-dermal, or intramuscular injection, or by inhalation, or sub-lingual administration.
[0026] Further disclosed herein is a method of immunogenic composition preparation that is appropriate for flu immunogenic compositions. The immunogenic composition is designed to confer immediate and long-term protection against viral infection and is particularly useful against pandemic flu viruses. It is also designed to be an effective immunogenic composition by using prestocked components that can be easily formulated into a immunogenic composition and quickly deployed where needed.
The Influenza A Virus
[0027] The influenza A virus consists of a viral RNA core surrounded by a viral coat composed of multiple units of hemagglutinin (H) protein and multiple units of neraminidase (N) protein. There are 16 known varieties of H protein and 9 known varieties of the N protein. These are designated as H1 thru H16, and N1 thru N9 respectively. Each strain of flu virus has one H component and one N component making up its viral coat. For example, one avian flu virus is composed of H5N1 one mild strain of human flu was found to be H9N and a virulent strain of human flu was found to be H1N1.
[0028] When different strains of flu virus co-infect the same host they may exchange genetic information resulting in a reshuffling of the H and N components to form a new viral strain. For example, if the flu strain H5N1 infects a human who is also infected with a flu virus such as H1N2, it is possible that a new flu strain H5N2 or a new flu strain H1N1 could be created. The new strain will exhibit a different infectivity and virulence profile from its parent strains.
[0029] Based on the number of different H antigens and N antigens exhibited by the known influenza A viruses, the total number of permutations that could theoretically result from genetic reshuffling is calculated by multiplying the 16 H varieties by the 9 N varieties to give 144 possible flu strains. In addition there are minor antigenic variations (i.e. clades) that will result in even greater antigenic diversity.
[0030] This application discloses that it would be more advantageous to stockpile individual components of the immunogenic composition and to then assemble the final immunogenic composition once the flu strain was identified rather than trying to develop a complete immunogenic composition against a predicted flu strain. This approach eliminates the time required to grow the predicted virus strain and produce large quantities of the immunogenic composition. It also provides a more efficient method of ensuring that the immunogenic composition is specific for the virus. For example, many companies are stockpiling vaccines prepared against the avian flu virus H5N1 because of the possibility that the H5N1 virus strain could become pandemic. But if there was a genetic shift and the pandemic flu virus turned out to be a H1N1 strain instead, the H5N1 immunogenic composition would not provide immunity against the H1N1 virus. However, if there were stockpiles of the H1 antigen and the N1 antigen as disclosed herein, then a multivalent immunogenic composition composed of a H1 univalent immunogenic composition and N1 univalent immunogenic composition would be available and expected to provide better protection as they would be targeted against the appropriate strains causing the virus. Further, the stockpiled immunogenic composition components can be assembled very quickly and be deployed in an effective time to where they are needed.
[0031] It should also be noted that there are antigenic variations within each type of isolated influenza A strain. For example, the H5 antigen of the H5N1 virus will show antigenic differences between different H5N1 isolates. In developing an immunogenic composition those H5 antigenic subtypes are selected which show the best cross-reactivity with other H5 isolates and/or develop several immunogenic compositions directed against selected antigenic sub-types. This also applies for other influenza A strains that exhibit antigen variation within the strain.
[0032] In another embodiment of this disclosure, the immunogenic composition also includes a purified antigen prepared from the conserved matrix (M1 and M2) regions of the flu virus coat.
[0033] In order to obtain the best immune response the viral antigens are utilized in a purified and isolated form and each viral antigen is presented separately to the antigen processing and/or immunocompetent cells. In this manner each clone of immunocompetent cells is programmed to react to only a particular antigen.
[0034] In order to obtain optimum immunity against a particular flu strain several of these univalent immunogenic compositions directed against different antigens of that strain are used to vaccinate the individual. For convenience and efficiency, the selected univalent immunogenic compositions are optionally combined to form a multivalent immunogenic composition prior to vaccination.
Viral Antigen
[0035] In order to prepare a viral antigen, the different H, N and M viral antigens are purified from viral cultures. In one embodiment, the antigens are prepared from cultured viruses. First, a selected strain of influenza A virus is used to inoculate embryonated eggs or cell cultures. After a period of multiplication the virus is extracted from the culture medium and purified using standard production techniques such as chromatography. After inactivation, the viral antigens are separated from other viral components using standard purification methods. The method of culturing the virus and the preparation of purified viral protein is known to those skilled in the art. The methods of antigen purification such as gel-filtration, affinity chromatography and other methods are known to those skilled in the art.
[0036] In another embodiment, the purified antigens are prepared by genetic engineering techniques. The genetic code of the particular viral antigen is incorporated into the genomic DNA of a transfected host cell. And the transfected cell is induced to express the corresponding H or N or M viral protein. Non-limiting examples of host cells suitable for production of the viral antigens disclosed herein include bacteria, yeast, insects, mammalian, or plant cells. For example, if the H1 viral antigen was selected, then the genetic code for H1 would be prepared and used to transform a host cell and the H1 antigen is produced as a recombinant protein. The expression level of the recombinant protein is increased by the use of suitable promoter genes. The recombinant antigen is purified by downstream processing and prepared for long term storage by lyophilization, or freezing or other similar processes. The method of genetic engineering and downstream processing are known to those skilled in the art.
[0037] In one embodiment, the antigens are prepared using the Baculovirus vector expression system in insect cells or larvae of the silkworm Bombyx mori. In this system the genetic code for the viral antigen is linked to the polyhedron promoter gene and used to produce a transformed baculovirus. When the transformed baculovirus is used to infect Bombix mori cells they are induced to express the recombinant viral antigen. Similarly, silkworm larvae infected with the transformed baculovirus are induced to produce large amounts of viral antigen in their hemolymph. This method of baculovirus expression of recombinant proteins and downstream processing is known to those skilled in the art. The use of silkworms as inexpensive "bioreactors" to produce large quantities of viral antigen is especially suited for developing countries that do not have access to the advanced and expensive vaccine production capacity of industrialized nations.
[0038] The viral antigen is isolated and substantially purified such that only the desired antigen is present in the composition, excluding undesirable antigens, proteins, lipids, or carbohydrates present in the starting material. For the purposes of the present disclosure, the term antigen includes full length proteins, and epitopes or fragments of the full length proteins which maintain the appropriate immunogenicity or ability to stimulate an immune response.
[0039] This application discloses the inclusion of a polynucleotide into the immunogenic composition. Polynucleotides are molecular chains of nucleotides usually deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). They can be of cellular or viral origin or they can be synthesized. When administered to an individual the polynucleotide has two important functions. First, it has an immune stimulating effect, and second it stimulates the production of interferons which are compounds known to have an inhibitory effect upon viral infections. For maximum anti-viral effect the polynucleotide is typically double-stranded DNA (dsDNA) or double stranded RNA (dsRNA).
[0040] Interferons belong to the large class of glycoproteins known as cytokines. They are natural proteins produced by the cells of the immune system in response to challenges by foreign agents such as viruses, parasites and tumor cells. Interferons are produced by a wide variety of cells in response to the presence of double-stranded RNA, a key indicator of viral infection. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection. When the antigen is presented to matching T and B cells, those cells multiply, attack and degrade the infectious agent. As disclosed herein presenting the polynucleotide simultaneously with the antigen to the same antigen processing or immunocompetent cell potentiates the immune response in addition to inducing the production of interferons.
[0041] In one embodiment of this disclosure a natural dsRNA polynucleotide extracted from any number of known viral or bacterial agents is used. Such agents include influenza A virus, influenza B virus, Sendai virus, E. coli etc. The methods of extraction, amplification using polymerase chain reaction (PCR), and purification of the natural polynucleotide are known to those of skill in the art. A synthetic polynucleotide can also be used. According to this disclosure exemplary synthetic polynucleotides are double stranded nucleic acids selected from the group consisting of: polyinosinic acid and polycytidylic acid (poly-IC), polyadenylic acid and polyuridylic acid (poly-AU), polyinosinic acid analogue and polycytidylic acid, polyinosinic acid and polycytidylic acid analogue, polyinosinic acid analogue and polycytidylic acid analogue, polyadenylic acid analogue and polyuridylic acid, polyadenylic acid and polyuridylic acid analogue, and polyadenylic acid analogue and polyuridylic acid analogue.
[0042] The polynucleotide chain may be modified by substituting other bases into the chain at specified intervals or by attaching additional compounds such as poly-L-lysine carboxymethylcellulose to the nucleotide chain. For example poly-IC can be stabilized by adding poly-L-lysine to form a new polynucleotide termed poly-ICLC. Poly-ICLC has shown anti-viral protection against a variety of viruses including vaccinia, hepatitis, influenza, herpesvirus, rabies, Japanese encephalitis, West Nile virus, Ebola virus, and HIV. Of particular note is that intranasal poly-ICLC administration can protect mice for as long as 3 weeks from an otherwise lethal dose of influenza virus.
[0043] In preparing the univalent immunogenic compositions of the present disclosure, the polynucleotide is mixed with the viral antigen so that both moieties are presented simultaneously to the same antigen processing and/or immunocompetent cell. The ratio of antigen to polynucleotide used in the univalent immunogenic composition may range from the order of 100:1 to 1:100. Depending on its potency the total amount of viral antigen used per univalent immunogenic composition is expected to be within the range of 1 μg to 100 μg per dose.
[0044] In aspects of this embodiment, an immunogenic composition disclosed herein may include a dose of viral antigen that is, e.g., 90-100 μg, 80-90 μg, 70-80 μg, 60-70 μg, 50-60 μg, 40-50 μg, 30-40 μg, 20-30 μg, 10-20, or less than 10 μg, less than 20 μg, less than 30 μg, less than 40 μg, less than 50 μg, less than 60 μg, less than 70 μg, less than 80 μg, less than 90 μg, or less than 100 μg. In other embodiments, the ratio of antigen to polynucleotide in the univalent immunogenic composition is, e.g., 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70. 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100.
Immunogenic Composition
[0045] The immunogenic composition is prepared by combining a viral antigen and polynucleotide and assembling them into a nanoparticle or liposome. To prepare the immunogenic composition, the viral antigen and polynucleotide are bound to the surface of a solid particle matrix such as colloidal gold particles, chitin nanoparticles, or other similar nanoparticles. The immunogenic composition components may also be incorporated into solid lipid nanoparticles. The methods of preparing nanoparticles include precipitation, homogenization and crystallization methods and are known to those skilled in the art.
[0046] In another embodiment, the coated nanoparticles are administered in conjunction with an adjuvant to enhance their uptake by antigen processing cells. Additionally, the coated nanoparticles may be incorporated into an oil-in-water emulsion which has a known adjuvant effect. Suitable adjuvants are known to persons of ordinary skill in the art.
[0047] The immunogenic composition may also be prepared in the form of liposomes which are composed of an aqueous core and an outer lipid bilayer membrane. Liposomes are prepared using different lipid compositions. For example, they are prepared using a mixture of one or more of the following compounds: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinsitol, monosialoganglioside, sphingomyelin, distearoylphosphatidylethanolamine, distearoylphosphatidylcholine, dimyristoyl-phosphatidylchol ine, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylcholine and the like. Cholesterol is also often included to stabilize the bilayer lipid membrane. The mixed lipids are dissolved in a solvent such as chloroform and dried under vacuum to form a lipid film. The dried lipid film is hydrated in a solution in which the viral antigen and polynucleotide are dissolved. The hydrated lipids will form liposomes entrapping the viral antigen and polynucleotide within the aqueous center of the liposome. Depending on the mixture of lipids used to prepare the liposomes and the method employed, such as freeze/thawing or homogenization or sonication, the liposomes formed may be multilamella or unilamella, and they may range in size from several thousand nanometers in diameter to below one hundred nanometers in diameter. The methods of preparing liposomes are known to those skilled in the art.
[0048] In another embodiment of this disclosure, a lipid formulation mixture is employed that will spontaneously form liposomes when mixed with an aqueous solution. The methods of manufacturing self-assembling liposomes are known to those skilled in the art.
[0049] In yet another embodiment of this disclosure the outer layer of liposomes may incorporate a known lipid adjuvant such as monophosphoryl lipid A (MPL). The basic liposome formulation will consist of a phospholipid compound such as phosphatidylcholine, cholesterol and MPL.
[0050] In another embodiment, a self-assembling liposome formulation incorporates an immune stimulating polysaccharide compound such as pullulans. The pullulans compound is anchored into the liposome membrane layer and stimulates uptake by the dendritic cells of the immune system. The dendritic cells are antigen processing cells responsible for initiating the immune response.
[0051] Also within the scope of the present disclosure are kits containing one or more univalent immunogenic compositions, the components for one or more immunogenic compositions, and instructions for use.
[0052] In one embodiment, the kit contains two, three, four or more isolated and purified viral antigens, polynucleotides and reagents to prepare two, three, four or more univalent immunogenic compositions. In some embodiments, the kits also contain an appropriate adjuvant.
[0053] In another embodiment, the kit contains two, three, four or more univalent immunogenic compositions. In some embodiments, the kits also contain an appropriate adjuvant.
[0054] Vaccine Administration
[0055] The mode of administration of the immunogenic composition may be by a route including scarification, or by injection, or by inhalation. In one embodiment, each univalent immunogenic composition is administered separately so that the antigen processing cells upon arrival at each site of administration will become programmed to develop immunity to only that particular antigen. In another embodiment, multiple univalent immunogenic compositions are administered in combination or simultaneously injected at different locations.
[0056] In order to obtain broader immunity to a viral strain it may be desirable to vaccinate with multiple viral antigens of that strain. For example, if the viral strain was avian flu virus with the H5N1 antigenic makeup, the vaccination program would administer two or more immunogenic compositions given to the individual. One immunogenic composition incorporates the recombinant H5 antigen plus a polynucleotide, a second immunogenic composition incorporates the recombinant N1 antigen plus the polynucleotide, while a third immunogenic composition incorporates the recombinant M antigen plus the polynucleotide. The individual injection sites may be placed close to one another (e.g. using a multineedle injector) or they may be spaced widely apart (e.g. on different arms); or be given at different times (e.g. on different days). The individual immunogenic compositions may also be mixed to prepare a multivalent immunogenic composition and diluted sufficiently so that each antigen processing cell is predominantly exposed to only one antigen. The same rationale for preparing tailor-made immunogenic compositions would apply to other influenza strains whose antigenic identity is known. Similarly for inhalation or sub-lingual administration, each immunogenic composition preparation can be given at different times or on different days.
Example of a Vaccine to Treat Pandemics
[0057] For illustrative purposes the preparation of a model immunogenic composition to treat an influenza A pandemic is disclosed herein.
[0058] The first step is to identify the strains of influenza A virus that pose the greatest threat of causing a pandemic. These will be the viral strains that are known to infect humans. Specifically, the H, N, and M subtype of the virus is determined.
[0059] The hemagglutinin (H) antigen is responsible for the first phase of infection which is the attachment of the virus to the cell; while the neuraminidase (N) antigen is responsible for the last phase of infection which is to cleave and release the viral particle from the infected cell. Therefore an immunogenic composition prepared against the H antigen is more effective in preventing viral infection, while a immunogenic composition prepared against the N antigen is more effective in limiting the transmission of the virus from host to host.
[0060] Based on this reasoning the most important antigens for preparing the immunogenic composition will be the hemagglutinin antigen sub-types that show the highest virulence and capacity for human to human transmission.
[0061] To increase host immunity to infection and to limit viral spread, immunogenic compositions against the N antigens from viral strains known to infect humans are also included.
[0062] Again, for illustrative purposes only, the preparation of a model immunogenic composition against the avian flu virus using selected embodiments is disclosed herein. For an avian flu virus with an H5N1 antigenic makeup, two univalent immunogenic compositions are prepared. One immunogenic composition is directed against the H5 antigen subtype and the other immunogenic composition against the N1 antigen. In this example, the recombinant H5 and N1 proteins are expressed in silkworm larvae using the baculovirus expression system. Briefly, the gene for the H5 protein is in-frame fused with the polyhedrin (Ph) gene under the control of Ph promoter and used to co-transfect cultured BmN cells with modified linearized Bombyx mori baculovirus DNA to produce recombinant virus (rBacPh-H5). The recombinant rBacPh-H5 virus is used to infect silkworm larvae which are induced to produce large quantities of recombinant H5 protein in the hemolymph. Similarly, the gene for the N1 protein is in-frame fused with the gene under the control of Ph promoter and used to co-transfect cultured BmN cells with modified linearized Bombyx mori baculovirus DNA to produce recombinant virus (rBacPh-N1). The recombinant rBacPh-N1 virus is used to infect silkworm larvae which are induced to produce large quantities of recombinant N1 protein in the hemolymph. The recombinant H5 and N1 proteins are purified using conventional laboratory techniques and stored lyophilized until required.
[0063] In this embodiment example, the polynucleotide used in the univalent immunogenic composition is the synthetic polynucleotide poly-ICLC which is a potent immunomodulating agent. The methods of preparing synthetic polynucleotides are known to those skilled in the art. The polypeptide chain can be prepared in bulk and stored in a suitable manner until required.
[0064] In this example, a self-assembling liposome incorporating the adjuvant compound is selected to prepare the univalent immunogenic composition. The liposome components such as phosphatidylcholine, cholesterol and adjuvant are premixed in bulk, vacuum-dried and stored frozen until required. Upon addition of an aqueous solution of the premixed viral antigen and polynucleotide the hydrated lipids will spontaneously form liposomes enclosing the antigen and polynucleotide within. The formed liposomes are lyophilized with a cryoprotectant agent and stored in bulk until required.
Other Applications
[0065] The concept of stockpiling immunogenic composition components can also be applied to seasonal flu where a univalent immunogenic composition prepared against the influenza B virus can be combined with the univalent immunogenic compositions prepared against the influenza A virus strains to give broad protection against developing the flu.
[0066] Disclosed herein is an integrated approach to developing an effective immunogenic composition for pandemic influenza and other viruses capable of causing a pandemic. The elements consist of: 1) producing and stockpiling recombinant viral antigens; 2) producing and stockpiling a selected double-stranded polynucleotide; 3) producing and stockpiling materials to make nanoparticles or liposomes; 4) assembling the recombinant viral antigen and polynucleotide into a univalent immunogenic composition; 5) administering different univalent immunogenic compositions to elicit a sustained immune response; and 6) combining different univalent immunogenic compositions to make a multivalent immunogenic composition to facilitate mass vaccination.
[0067] The same general principles enumerated here can be applied to preparing immunogenic compositions against a variety of other viral and bacterial pathogens. There are many known infectious pathogens that are capable of causing pandemics with significant morbidity. The WHO Epidemic and Pandemic Alert and Response list includes: anthrax, avian influenza, Crimean-Congo haemorrhagic fever, dengue haemorrhagic fever, Ebola haemorrhagic fever, hepatitis, influenza, Lassa fever, Marburg haemorrhagic fever, meningococcal disease, plague, rift valley fever, severe acute respiratory syndrome (SARS), smallpox, tularaemia, and yellow fever.
[0068] There is also the threat of bioterrorism wherein infectious agents can be modified to make them more infectious and virulent. The most effective means to prepare for these threats is to develop a vaccination program that is effective, safe and can be rapidly deployed.
[0069] The above descriptions are given by way of example, and not limitation. Given the above disclosures, one skilled in the art could devise variations that are within the scope and spirit of the invention disclosed herein. Further, the various features of the embodiments disclosed herein can be used alone, or in varying combinations with each other and are not intended to be limited to the specific combination described herein. Thus, the scope of the claims is not to be limited by the illustrated embodiments described in this invention.
[0070] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0071] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0072] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[0073] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[0074] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.
[0075] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.
[0076] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
Patent applications by Henry J. Smith, Temecula, CA US
Patent applications in class Liposomes
Patent applications in all subclasses Liposomes
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- 自治州 : Principality of Asturias
On the Asturian coast the anglerfish, a fish with firm, tasty flesh, is known as the 'pixín'.
The anglerfish is a teleostean coliform fish with a large head and enormous mouth, very consistent, without scales, and with white flesh that is very tasty and low in fat. Because of the way it looks, it is also known as the 'toad fish' or 'pejesapo'. Its head is used to make soups and its tail offers exquisite pieces. It is the base for a number of recipes in Asturias: lobster-style anglerfish, 'a la sidra' (in cider sauce), 'fabada con pixín' (bean stew with anglerfish', wild mushrooms with anglerfish or fried anglerfish, among others.
- 産品の種類: Fish
- 産地: The anglerfish, fished off the coast of Asturias, is of an excellent quality.
- お勧めの飲み物: Crianza and non-crianza White Wines.
- 購入場所: 'Pixín' is available at most Fish Exchanges at the ports along the Asturian Coast, as well as in many fish markets throughout the province.
- レシピ: Fish soup
- Energy: Low
- Cholesterol: Low
- 注意: Rich in Proteins.


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iPhone is one of the most popular electronic gadget and mobile phone. Tt has created a revolution in mobile market people it is popular due to its sleek design and quality graphics. This PSD is very useful for mobile developers and designers who need a professional way to show mock-ups to clients or to work out ideas. Check it Out.
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Langauge: English
Sex: MALE
Rating: 7
Category: Gay
i was going home on the bus from school and a guy sitting next to me was listening to some pretty cool music which i recognised but i couldnt remember the band. So i started a conversation with him about it and he introduced himself as sean.He told me how big a fan he was and how much he was into them. He said that he had all their cd's at his house if i wanted to borrow them and he lived pretty close to me so i walked back to his place. His house was small but he had heaps of cd's by really cool bands and i was really impressed and i think he spotted my adulation. He then told me straight out that he was gay, but i didnt have any problem with it and i was a little curious! I just asked him about being gay and what it was like, but nothing he said really shocked me, and i was getting turned on when he started talking about sexual stuff. He spotted me shifting around on the couch and came over and sat next to me.He asked me if i liked him and i said that he was quite good looking and very funny, (in reality i thought he was a hunk!). He then lightly leaned forward and kissed me lightly on my lips, and pulled back to see my reaction. I was stunned for a but then i gave hime a little smile and leaned over and kissed him back, all very gentle and slow. We just kissed for a while before he suggested we have a shower. I was really curious and horny by now so we got undressed in front of each other and got into the shower. Both of us had fairly average bodies and cocks but i thought he looked very sexy and we just ran the shower and kissed in its warmth. Being very new to sex then i got the biggest boner straight away and it was poking him but he just laughed. I asked him if i could just have a close look at his body as it was a new thing to me being with a guy. He asked me just to soap him all over as he loved the feel so starting from the top and moving down i slowly rubbed his chest and arms, before giving him a light back massage. It was then that he suggested i finish the massage in the bedroom…hint, hint! well we got out and he lay face first on the bed naked as i began to oil his shoulders and back before moving down. I massaged his lower back and then moved to his legs, working my way up each leg. By now i was rock hard and as i massaged his upper leg i ran a finger under his balls, just touching them. He then turned over and was rock hard as well and he told me i was a great masseur. I then laid down and he did the same for me and i loved the feel of his hands.He then aked me to turn over and i did with my cock sticking out like a flagpole! He asked me if i wanted to keep going and i said of course! he then leant down and slowly licked my bellybutton before kissing down further, around the base of my cock, before taking my sack in his mouth.This was driving me crazy and i had precum seeping from my cock, as he slowly took my circumcised head in his mouth and swirled his tounge around. I was in heaven as he lowered his mouth over my cock and took all my 5″. I could feel my orgasm in about a minute and he kept sucking as i bucked my hips and shot into his mouth. He took it all and slowly sat up as i lay back in extacy. Straight away i laid him down and tried to reciprocate my pleasure. It was a very strange sensation holding his manhood in my mouth as i slowly sucked and caressed him back. He took muck longer and when he came I had to take my mouth off as he came over my throat and chest. I said sorry but he just lay back and said i was fantastic, naturally i was very happy. We just lay next to each other for about half an hour afterwards, caressing each others bodies but i never went any further than that and he was more than happy to have it like that. Many times after school i went over and played around with sean but he moved city later and I havent seen him since, but he does email me and say hello quite often. He was the only guy i ever slept with and i now have a beautiful girlfriend who has shown me everything about making love.


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Panagiotis Spyridakos
PhD Researcher
Phone: 33842
Room: Driving SIM 238
Email: firstname.lastname@example.com_
Research Group: Human Factors and Safety
Key Research Interests
- Human Machine Interface (HMI) evaluation in driving - effect of HMI tasks on safety and performance of the driver.
- Driver distraction modeling and simulation.
- MSc (Hons) in Computational Intelligence and Robotics, Automatic Control and Systems Engineering Department, University of Sheffield, 2014.
- BSc in Computer Science, Computer Science Department, Aristotle University of Thessaloniki, 2012.
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Welcome to Preferred Alliance, Inc. (PA), your third-party administrative solution for drug and alcohol testing compliance.
Whether you're considering a drug-testing policy and procedures, need to comply with mandatory Department of Transportation (DOT) regulations, or simply want the most current information on testing, we can help. With Preferred Alliance, you get decades of experience in drug and alcohol testing, a portfolio of end-to-end services and a 24/7 relationship. We can help you manage non-compliance risk and keep your workplace drug-free.
Can We Test?
Yes. Every employer has the right to ensure a drug and alcohol-free workplace. If you are not regulated by the DOT, we'll help you develop a comprehensive drug-testing program, guide you through its complexities and custom fit it to your needs. From constructing a drug test policy and procedures—your program's backbone—to ongoing maintenance and billing, Preferred Alliance can support every step you take.
Should We Test?
If you are mandated to test under DOT regulations, we'll review your written policy and procedures to ensure your program is current. Preferred Alliance takes a personal approach to every relationship, whether you are an individual owner-operator or a multinational. Consider us a one-stop shop that provides a custom—vs. one-size-fits-all—solution to your needs.
© Preferred Alliance, Inc. All rights reserved. All other copyrights are reserved by their respective holders.
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HE is the best there is at what he does.
Those are the words usually reserved for X-Men icon and comic book favourite, Wolverine. But in Logan, the character's swansong, that mantra is more fitting for actor Hugh Jackman.
The Australian star has played Wolverine nine times over 17 years in various films set in the X-Men universe. Some of those movies have been great, others have been big let-downs but the common thread has been Jackman's muscular – in more ways then one – performances
He has given his all to the character but perhaps never more so than now. Fans of Wolverine will be extremely satisfied with this brutal, bloody and bruised send-off to an icon.
Set in the near future and loosely based on Steve McNiven's Old Man Logan graphic novel, world weary Logan (Jackman) cares for an ailing Professor X (Patrick Stewart), who is now in his 90s.
In previous films, Logan – or Wolverine – has been more or less unbeatable with rapid healing abilities and a skeleton and retractable claws laced in a fictional indestructible metal called adamantium. But in James Mangold's movie you finally get to see a vulnerable side to the ageing character.
His healing ability has weakened and one of his claws no longer retracts properly. He spends his time attempting to hide from the world until a young girl called Laura (Dafne Keen) arrives who appears to be able to mimic his mutant abilities.
Pursued by a shadowy corporation, Laura brings out the reluctant hero and fiercely loyal protector in Logan. This is finally the Wolverine film that fans have been waiting for and credit also goes to studio 20th Century Fox for giving director James Mangold a free rein.
It is dark, intense and the finale packs a punch. It also does not hold back in terms of violence – how it got through the ratings process with just a 15 is hard to fathom.
Newcomer Dafne Keen is a revelation as Laura, bringing out the insecurity of a young girl and the ferocity of a cornered animal. Patrick Stewart is on predictably top form too as the former leader of the X-Men, Charles Xavier.
The deteriorating professor's telepathic mind, classed as a weapon of mass destruction, is breaking which raises the stakes in the story even more.
In a world of constant sequels and spin-offs, this standalone tale satisfyingly brings closure to a beloved character.


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Welcome to Wethersfield Public Schools
Our system encompasses seven schools which provide a high academic standard of education to approximately 3,700 students in PreK through grade 12. Located in historic Wethersfield, Connecticut, the District prides itself on its record of student achievement and academic standards.
Our focus is on ensuring that students acquire the skills and knowledge for lifelong learning, develop a positive sense of self and self-discipline, and function as responsible citizens of society.
Our students benefit from a close partnership between the District and the Wethersfield community as well as from strong parental support and involvement. More than 3,600 students attend one of five neighborhood K-6 elementary schools, matriculating to our middle school and high school. At all levels, students are exposed to a variety of learning experiences both in the classroom and through athletics, the arts, and other co-curricular activities - experiences that help them explore and discover their talents and interests. Students are guided in this journey of discovery by our talented and dedicated faculty who are committed to all learners and whose mission is to ensure that all students truly can be successful.
Holidays of Wethersfield's World
Wethersfield's rich diversity is on display this summer, as parent volunteers from Wethersfield's People Empowering People teach up with Wethersfield Historical Society to present an exhibit to celebrate diversity of local families.
Dollars for Scholars Golf Outing
Come support the 21st Annual Wethersfield Dollars for Scholars Golf Outing (co-sponsored by Wethersfield Country Club) on Monday, August 6, 2018. For more information, contact Gina Cusano (firstname.lastname@example.com).
WPS Teacher and Para Educator of the Year
It is with great excitement that Central Office is pleased to announce the 2018-19 Teacher of the Year and Paraeducator of the Year. Jennifer Lizee-Hammer, Literacy Curriculum Specialist at Webb and Emerson-Williams Schools and Pat Mahder, Para Educator at Hanmer School were surprised with this great honor today!


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There are many preservatives used in food to help it stay fresh including aspartame, sodium nitrate, monosodium glutamate, high fructose corn syrup and BHA. The preservatives help to maintain taste and appearance.Continue Reading
Most additives in food have no adverse effects from eating them, but it is nice to know what is being ingested. Some of the chemical preservatives used in food are:


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For paid members only, here's a coupon code that will allow you to watch a full Photoshop course of mine for free. The course is...
My classes on creativeLIVE
I have some classes on Kelby Training
Looking for training on Photoshop CC? Try learningphotoshop.cc
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( Originally Published Early 1900's )
The name vegetable means anything that grows in the earth, but is now commonly applied to those plants which are used for food. Vegetables may be classified as flowers, seeds, roots, and tubers, according to the part that is eaten.
With the exception of the cereals, potatoes, and beans, vegetables have but little actual food value, but, like fruits, they are important in the diet on account of their salts and acids and for the cellulose which furnishes the bulk needed to keep the digestive machinery working. A diet low in green vegetables or fresh fruits is apt to cause constipation.
All vegetables are largely composed of water ; next to water, carbohydrates are most abundant ; traces of protein and fats are also found in them.
Mention some vegetables that contain fats (see Table). One that contains protein. One that contains starch. One that contains sugar. Test vegetables for these elements. (See Experiments 1-. Classify vegetables named in recipes of this chapter (including dried beans and potatoes) as green and starchy, according to compositio.
By a simple experiment let us see what we mean by cellulose.
EXPERIMENT I. Grate a turnip. Squeeze the pulp into a bit of cheese-cloth or a fine sieve and then inspect it carefully. The woody fiber remaining is celinlose. It must be softened by heat and moisture before it can be used and even then the body will get little actual nutriment from it. The more cellulose in a vegetable the more need there is of cooking it.
EXPERIMENT II. Prepare a potato in the same way as you prepared the turnip. Inspect the pulp and test it for starch. (See Experiment I, page 43.) The starch is so closely connected with the fiber that it cannot be separated except by cooking.
EXPERIMENT III. Grate a carrot. Squeeze out the juice, put it into a small saucepan, and boil it down. Taste it. What do carrots contain 1 What other root vegetables contain this substance?
EXPERIMENT IV. Examine thin slices of vegetables under a microscope, noticing the fiber.
EXPERIMENT V. Pare a potato and weigh it. Cut it in thin slices and put them into a warm, dry place (the warming oven or the back of the range will do) where they will dry but not cook. When thoroughly dry (several days may be needed) weigh them. What per cent has been lost? What substance was lost? Dry lettuce in this way, noting per cent of water.
Retaining the Minerals. The value to the body of the mineral salts derived from vegetables has only lately begun to attract the attention of physiologists. It has been proved beyond a doubt that perfect nourishment is possible only when all the required elements are supplied. Green vegetables are one of the chief sources of potash, iron, and other needed minerals. If much water is used in their cooking, not only is flavor lost but also these valuable salts. When cooked without paring, vegetables retain most of these salts, as do those whose juice is used as a sauce or those cooked in such a way that all the water used is evaporated. Where possible, cook vegetables by steaming or in a very small quantity of water.
Softening the Cellulose. As was seen from Experiment I, vegetables contain a tough woody fiber that requires softening by cooking. Some of this fiber is so tough that it must be removed either before or after cooking ; for example, the husks of corn, the skin of beets, carrots, tomatoes, and the shell of the pumpkin. Some vegetables are cut into small pieces so that they may be softened. (Mention one.) The cellulose in young vegetables is more tender than in old ones ; therefore they are more easily cooked. One of the important points in cooking vegetables is to soften the woody fiber. Vinegar is served with turnips and beets, and helps to soften the woody fiber.
Preserving the Flavor. Another important point in the cooking of vegetables is to preserve the flavor. It was found, that the carrot juice was sweet because of the sugar. If the carrots were cooked in a large quantity of water, the juice would be in the water instead of in the carrots. Suggest a plan for cooking them so that the juice will remain.
As all vegetable flavors are easily dissipated by great heat, vegetables should not be boiled too rapidly. Unless very highly flavored, they should be cooked in the least water possible and drained, if needed, as soon as tender. The kind of water in which vegetables are cooked has much to do with their softness and flavor. Soft water, such as cistern water, should have a tea-spoonful of salt added to each gallon when used for cooking fresh vegetables. In the case of dried peas or beans, the Salt should be added after they are cooked.
Hard water may be softened by adding half a teaspoonful of soda to each gallon of water. This is needed for both fresh and dried peas and beans. Other green vegetables do not need soda, as their color and flavor are best preserved by slightly hard water.
Never cook vegetables in an iron vessel. Perfectly smooth enameled ware is best. Use freshly boiled water.
EXPERIMENT VI. Cook green vegetables, covered and uncovered, and note difference in color and flavor.
Seasoning and Serving. Do not serve more than one semi-liquid vegetable at one time. For instance, do not serve stewed tomatoes and stewed corn at the same meal ; if it is desired to have the corn stewed, serve the tomatoes sliced, or have the corn cooked in a pudding or boiled on the cob if the tomatoes are stewed.
Any vegetable which is not served in the water in which it is cooked must be very thoroughly drained. Turn the vegetable into a colander or sieve, let it stand for a few moments, and then turn into the saucepan in which it was cooked and shake over the fire until quite dry. Greens, such as spinach or mustard, should always be pressed to remove the water, before they are brought to the table.
After draining, season so delicately that the flavor of the vegetable is not hidden; for instance, black pepper in green peas hides the flavor of the peas. For most vegetables, melted butter or a simple sauce is used. Allow two tablespoonfuls of melted butter or one cup of thick white sauce (see page 118) to each pint of vegetables. Turnips and beans may be cooked with a little salt pork. Corn, tomatoes, and green peas require a little sugar. Beets and greens are improved by a little lemon juice or vinegar. Serve cooked vegetables in warm dishes. All vegetables should be dry enough to be served on the plate and eaten with a fork.
Selection and Care. Vegetables are cheapest and best when in season, that is, when grown near the marketing place. Wilted vegetables are tough and lack flavor.
Root vegetables should be smooth and tender and of medium size, the tops being fresh and green. All green vegetables should be, fresh and crisp and not too coarse. Vegetables should be kept in a clean, cool, dry place.
Preparation for Cooking. Examine all vegetables carefully to see that there are no insects on them. Wash thoroughly as they may be sandy or may have poisons on them which have been used to destroy insects. Then pare and cut them in pieces if required.
Select canned vegetables very carefully. Buy reliable brands in cans which have the name of the packer on the can. Read the label carefully and see that colorings and preservatives are not used. Examine the cans; if they bulge, the contents are spoiled and gas has formed. Old canned goods are not safe, as the tin may have been corroded by the acid of the contents. If many canned goods are used, buy them by the case at the season when the freshly packed article has come into market (usually in the autumn), and store in a cool place. Fresh green vegetables are better and cheaper than canned ones at most seasons. Compare prices of home canned vegetables with prices of the factory product.
Cooking Canned Vegetables. As they have been cooked, canned vegetables need only to be reheated and seasoned. As soon as the can is opened turn the contents into a bowl and air for an hour. If the commercial canned goods are used, drain off the liquid from asparagus, peas, and like vegetables, and wash them in a colander.


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The origin of the name comes from Helen Porter Mitchell (1861 - 1931) - who had the stage name of Dame Nillie Melba. Her last name comes from her native city of Melbourne. It is believed that Auguste Escoffier (a well known chef of the time) is thought to have had a hand in both the creation of Melba toast and peach Melba.
Melba toast itself was a primary part of Dame Melba's diet in 1897. In 1897, Dame Melba was seriously ill and bland food was necessary. Another version of the story is that the toast was part of her constant struggle to lose weight. Melba toast entered common use in 1925 and was first mass-produced by the Devon Bakery in New York City.
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On January 26, a few of us performed over at St. Hilda's and St. Hugh's School as part of their Lunar New Year celebration. It was a fun experience for both our members and the students as well, as they gathered around us and wanted to play the drums after the performance!
Saturday, February 4, 2017
Celebrating Lunar New Year at St. Hilda's and St. Hugh's School
Posted by LP Larrabee at 10:25 AM
Labels: performance, St. Hilda and St. Hugh


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Tropical Custom Boat Names. Standard Size 3" tall x 24". Available in other sizesand fonts. Select fonts from font pop up selection. Custom orders please call 951-245-7127 or email us email@example.com A great place to look for fonts is dafont.com. Let us know font and WE CAN SEND A PROOF WITHIN 24 HOURS, print, cut and ship.
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John Boehner visited Mexico on Friday along with other members of U.S. Congress to address issues of security, economic and political ties with Mexican authorities
President Felipe Calderon urged the Lower House leader John Boehner U.S. to end arms trafficking that has fueled the drug violence in Mexico. A statement from the president stressed "the importance of implementing stricter measures to stop illegal trafficking of weapons and cash into our country."
Drug cartels in Mexico import most of their weapons from the United States.
Boehner and the Michigan legislator, Dave Camp, visited Mexico on Friday along with other members of Congress to address security issues, economic and political ties with Mexican authorities. The statement added that the U.S. delegation recognized the five-year offensive against drug cartels. The U.S. delegation visited Colombia and Brazil advance.


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Showcase of Products & Manufacturers
CuVerro is proud of our affiliation with leading manufacturers of furnishings and equipment who envision the benefit continuously clean surfaces provides. Our manufacturers are transforming categories with a range of products made with CuVerro® bactericidal copper alloys that are beautifully effective at killing bacteria1.
A CuVerro affiliation provides a true competitive advantage in the added value of a synergistic and collaborative supplier network, all with full support from Olin Brass. The Olin Brass affiliation provides the technical, R&D, distribution, and customer service support that customers require, as well as assistance with the regulatory compliance necessary to sell products made with bactericidal copper into the marketplace.
Launching a successful product requires more than an exceptional material CuVerro® provides the strength, performance, and clean look that high-touch surfaces demand. CuVerro® has been developed to provide component and equipment manufacturers with the exact materials for their specific applications.
Interested in learning more about products made with CuVerro? Click here
Interested in learning more about becoming a CuVerro manufacturer? Click here


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Action Potentials & Fibromyalgia
I have been reading a lot about fibromyalgia lately and in a book by a Dr. Amand, as reported by a couple of Drs. 25 years ago - states that calcium remains immobile due to a lack of Action potentials because people with FMS have a 25% drop of ATP levels in their muscle thus not allowing calcium to be moved via active transport. The Ca, providing a constant request for action doesn't allow time for the cells to rest and regenerate, resulting in overtaxed systems and resulting in everything from brittle nails to cerebral, musculoskeletal, gastrointestinal, and genitourinary problems. Trying to figure this out.© BrainMass Inc. brainmass.com August 21, 2018, 1:41 am ad1c9bdddf
Solution Preview
Hi again!
And thanks for asking for me by name. I appreciate it.
Now I can see the context of your question. I assume you're talking about Dr. Amand's book entitled "What Your Doctor May Not Tell You About Fibromyalgia." Right?
Basically, what you have described is correct -- but this is a systemic physiological problem, not a minute biochemical issue.
When the body has an impaired ability to convert ADP + Pi (inorganic phosphate) into ATP, problems will develop or, in this case, get worse.
First, the tissues (in this case, muscle tissue) will have an insufficient amount of ATP. This has been verified ...
Solution Summary
Action potentials in patients with fibromyalgia are discussed.


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A 38 year old female (para 3, gravida 3), complains of persistent bilateral mammary gland pain associated with a "lumpy" feeling when she does a self- examination. She carries a family history of breast cancer in both her mother and older sister. A biopsy revealed the following image.
The most likely diagnosis is:
- Medullary carcinoma
- Lobular Carcinoma in situ
- Fibrocystic disease
- Ductal carcinoma in situ
Click here to see the whole slide image.
Click here for the comparative pathology.
Scroll below for answers.
Answer: 3, Fibrocystic disease.
Fibrocystic Disease (Condition) is a hormonally associated benign mammary disorder affecting about 60% of women. The hormonal changes during the menstrual cycle cause the mammary gland to feel swollen, painful and "lumpy". Following menopause the changes usually subside. However, in cases of PHTS and history, there is an increased risk for mammary malignancy. The solid nests have irregular slits which are considered benign. The cysts are apocrine cysts.
Miltenburg DM, Speights VO Jr. Benign breast disease. Obstet Gynecol Clin North Am. 2008 Jun;35(2):285-300, ix. Review. PubMed PMID: 18486842.
Santen RJ, Mansel R. Benign breast disorders. N Engl J Med. 2005 Jul 21;353(3):275-85. Review. PubMed PMID: 16034013.
Valea FA, Datz VL "Breast diseases: diagnosis and treatment of benign and malignant disease" In: Katz VL, Lentz GM Lobo RA, Gershenson DM, Eds. Comprehensive Gynecology 5th Ed Philadelphia, Pa Mosby Elsevier, 2007, Chapt 15.
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Safe Harbor Behavioral Health of UPMC Hamot, located in Erie, Pa., is seeking a child psychiatrist to join a well-established comprehensive behavioral health outpatient program. This person will join an established practice serving more than 6,500.
Safe Harbor provides a collaborative and team-oriented work environment with collegiality, nursing support, and an opportunity to participate in research projects with Western Psychiatric Institute and Clinic. A continuum of nursing, case management, therapy, addiction services, and crisis intervention services support with the delivery of quality care.
Candidates should possess:
This position is available immediately. As an employee, you will enjoy an excellent compensation package including full benefits and paid CME.
Safe Harbor Behavioral Health was founded in 1993 and has a primary focus in serving adults with serious and persistent mental illness and children with serious emotional disturbance. Safe Harbor has been recognized for its innovation in behavioral health programming including comprehensive crisis intervention services, hospital diversion programming, partnership with local law enforcement, and participating in published research studies that have been instrumental to the field of psychiatry. Safe Harbor's Crisis Services is a National Suicide Prevention Lifeline Center.
Katie Bool Shaffer
Physician Recruiter
Phone: 814-877-3327
Email: firstname.lastname@example.com
Affiliated with the University of Pittsburgh Schools of the Health Sciences |
Supplemental content provided by Healthwise, Incorporated. To learn more, visit www.healthwise.org
For help in finding a doctor or health service that suits your needs, call the UPMC Referral Service at 412-647-UPMC (8762) or 1-800-533-UPMC (8762). Select option 1.
UPMC is an equal opportunity employer. UPMC policy prohibits discrimination or harassment on the basis of race, color, religion, ancestry, national origin, age, sex, genetics, sexual orientation, marital status, familial status, disability, veteran status, or any other legally protected group status. Further, UPMC will continue to support and promote equal employment opportunity, human dignity, and racial, ethnic, and cultural diversity. This policy applies to admissions, employment, and access to and treatment in UPMC programs and activities. This commitment is made by UPMC in accordance with federal, state, and/or local laws and regulations.
Medical information made available on UPMC.com is not intended to be used as a substitute for professional medical advice, diagnosis, or treatment. You should not rely entirely on this information for your health care needs. Ask your own doctor or health care provider any specific medical questions that you have. Further, UPMC.com is not a tool to be used in the case of an emergency. If an emergency arises, you should seek appropriate emergency medical services.
Pittsburgh, PA, USA | UPMC.com


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First look at the Asus Pro61s
Today it´s once again time for a laptop review, Asus sent us a sample of their brand new Pro61s laptop. With features as a Cire T5850 cpu, N Wifi, 16 inch screen, and 4570 videocard this really seems to be a killer. We are going to look at the screen, performance, and battery time in this review so lets have a closer look at what we can squeeze out of it.
About Asus:
Check out their website, http://www.asus.com/ContentPage.aspx?Content_Type=AboutASUS&Content_Id=9
15.6″ (HD, Glare Type)
Intel Core 2 Duo T5850 (2.1G, L2:2M)
1.3MP Cam
320GB 5400rpm SATA HDD
DVD Super Multi
WLAN 802.11bgn
Vista Home Premium
6 cell battery
No Bag&Mouse
The product:
Please click on thumbnails to get bigger pictures
Asus No.1 in quality it says on the box, that is something we are going to look at in this review. Inside I found a manual , waranty card, and a s-video adapter.
Please click on thumbnails to get bigger pictures
Inside you will also find a bundle of cd´s with drivers and software. The manuals explain everything there is to know about this laptop. I actually found more manuals and cd´s in this laptop than any of the previous ones I have ever looked at.


text: I am 110 years old, dont i look great? My friends and I make all these skits, you can check us out on youtube and facebook too. Our "production" name is dankman500. Were all from a shitty small town called Havertown in Pennsylvania, right outside of Philadelphia. We really hope everyone digs these skits because we really do and we put a lot of time and work into them. If you like them, let me know and if you don't, then you don't have to be a faggot about it. Check out our facebook page http//www.facebook.com/pages/Dankman500/251815235968?ref=ts


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Adapted from a recent online discussion.
Dear Carolyn:
So my worst nightmare just came true: My religious-convert sister (RCS) told my gay sister (GS) that RCS and her husband and children will not ever meet GS's children (1-year-old twins). GS is still welcome to visit RCS and her family alone. RCS's rationale seems to be that her children are getting old enough to ask hard questions and she doesn't know what to tell them about their aunts and their IVF cousins.
I am devastated that our family apparently will never again be all together, not to mention incredibly angry and hurt. Our parents were never happy that GS is gay but managed to cope, and have include GS's wife in the family for 10 years, so they are also upset at RCS's announcement. I have not yet spoken to RCS, as this JUST happened.
What on Earth do I say and do? I can't participate in any family gathering to which GS and her family are not invited, but I also can't stand the thought of cutting all ties with RCS and her family, given that I strongly disagree with her choice of estrangement in the first place.
Instead of going at RCS with outrage guns blazing — or just going silent — try talking to her about the very narrowly defined issue of what she says to her young children.
I believe RCS is demonstrating breathtaking cowardice and I would be outraged, but sometimes with family it's helpful to get away from clashing principles (gasoline on a fire, usually) and try a more practical route.
RCS is going to have to tell her kids something about some actual gay people someday, since they're bound to run across some eventually. If what she plans to say is something she can't say to her sister's (or a child's) face, or can't imagine having her kids parrot back to GS and family — then shouldn't she reconsider her words? Ask her. A defensible belief is one that covers strangers and loved ones, adults and kids.
It's a long shot, but if she's willing to discuss it with you — again, just this narrow, what-do-I-tell-my-kids question — then you two just might be able to work out phrasing that isn't a cop-out the way estrangement is. Her "will not ever meet" edict is hyperbolic poo anyway, since the kids will decide for themselves whom they meet the moment they become adults.
Dear Carolyn:
I've been single for a long time, and one of my good female friends and I have been sort of teammates through the dating maelstrom, trading war stories and such. But now I've met someone. I'm unsure how to break the news to my friend — we've talked about how she's been feeling left behind as her friends couple up. I just can't imagine this being news she wants to hear.
Probably not, but it's not a race and you haven't crossed a finish line. Each of your lives could go in any number of unexpected directions from here. Tell her you're happy about this new person, but you've also enjoyed being single with her as a teammate — and of course may soon enjoy it again, because, who knows? No matter how things turn out, you can choose not to leave her behind.
Write to Tell Me About It, Style, 1150 15th St. NW, Washington, D.C. 20071, or firstname.lastname@example.com.