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Check Your Car's Air Conditioning System
View Single Post
Holly Goodhead
Join Date: Oct 2016
Posts: 3,243
Why do hybrids use high voltage A/C compressors?
Why do hybrids use high voltage A/C compressors?
Vehicles with full-hybrid technology use high voltage compressors that are not dependent on the running of the combustion engine. This makes for greater A/C comfort in such vehicles.
A vehicle's interior that has become overheated can now be cooled down to the desired temperature before the start of any journey by using a remote control. This stationary cooling process can only be brought about if sufficient battery capacity is available.
The compressor is controlled with the lowest possible output with the necessary A/C requirements being taken into account. In the high voltage compressors used today, the power is regulated by adjusting the rotary speed in steps of 50 min-1. It is therefore not necessary to have an internal power control.
Function and design
In contrast to the 'swash plate' principle, which is primarily used in the belt-driven compressor field, high voltage compressors use the 'scroll' principle to compress the refrigerant. The benefits are that the weight is reduced by approximately 20% and there is a reduction in the cylinder capacity of the same amount, whilst the output remains identical.
In order to generate the right amount of torque for the drive of the electric compressor, a DC voltage of over 200V is used.
Specialist subject
You should be aware that appropriate staff training is required for the servicing of hybrid vehicles, with further skills imperative for those who service and repair the complex thermal management systems found in hybrids. In Germany, for example, those employees who work on high voltage systems are obliged to attend an additional two-day course in order to qualify as an "Electrician for High Voltage Systems".
The course teaches the technician to recognise the risks when working on systems of this kind and also how to switch off all the current to the system for the duration of the work. It is prohibited for people who have not attended specific training courses to work on high voltage systems.
Also, tasks dealing with general servicing and repairs (such as work on exhaust systems, shock absorbers, oil changes and the changing of tyres) may only be carried out by employees who have attended the same course, in order to learn about the risks associated with these types of systems.
Tools of the trade
It is also essential to use tools that comply with the specifications provided by the manufacturer of the hybrid vehicle. During the A/C check and service, steps must be taken to ensure that the electric A/C compressors ARE NOT lubricated with standard PAG oils, as these don't have the necessary insulation properties. POE oil or a special type of PAG oil is normally used instead, as these have the required properties.
Consequently, A/C service units with an internal rinsing function and a separate fresh oil reservoir should be used for the A/C check and service in hybrid vehicles. This then ensures that any mixing of the various types of fresh oil is prevented.
This help topic is subject to changes without notification. The information within is carefully checked and considered to be correct. This information is an example of our investigations and findings and is not a definitive procedure. The Sixties Garage accepts no responsibility for inaccuracies. Each vehicle may be different and require unique test settings.
Last edited by Holly Goodhead; 19-04-2017 at
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subset: commoncrawl


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Breed Animal Farm gems hack
Discription: The tale starts on a far-off land in the skies, called Pet Ranch. We had an excellent hands on experience, the animals were very interactive. Pig farming has began to end up being prominent among livestock farmers in Nigeria in the current time. Opposite to the method of mating animals of different breeds, pure-blooded breeding aims to establish and maintain stable traits, that animals will pass to the next generation.
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Prepare to check out a magical 3D globe overhead loaded with cute animals and abundant farmland! They're social pets. However in this short article, we will be looking at the profit or organisation side of animals farming. The pets rebellion after Jones drinks so much he does neglect the animals. With Task Pet Farm: An Accidental Journey right into the Secret Globe of Farming as well as the Reality Regarding Our Food, I have learned that the Canadian meat market is much from wonderful.
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Records, as well as in danger breed checklists, from the Farm Animal Genetic Resources Committee. Numerous read more and use publications provide regulations established by international boards for standardized nomenclature of outbred rats as well as rabbits (Festing and also others 1972), inbred rats (Festing as well as Staats 1973; Gill 1984; NRC 1992a), inbred computer mice (International Board on Standardized Hereditary Classification for Mice 1981 a, b, c), and also transgenic animals (NRC 1992b).
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- North America
- United States
- Michigan
- South Shore Marina
- Search
Traveling Luck for South Shore Marina Michigan, United States
The timezone in South Shore Marina is America/Iqaluit
Morning Sunrise at 09:10 and Evening Sunset at 18:40. It's Dark
Rough GPS position Latitude. 42.7753°, Longitude. -86.1792° , Elevation. 177m
Morning Sunrise at 09:10 and Evening Sunset at 18:40. It's Dark
Rough GPS position Latitude. 42.7753°, Longitude. -86.1792° , Elevation. 177m
Weather near South Shore Marina Last report from Holland, Tulip City Airport, MI 9.6km awaylight snow mist Temperature: -4°C / 25°F
Wind: 8.1km/h East/Northeast
Cloud: Few at 800ft Solid Overcast at 1600ft
Satellite map of South Shore Marina and it's surroudings...
Geographic features & Photographs around South Shore Marina in Michigan, United States
school building(s) where instruction in one or more branches of knowledge takes place.
- Harrington School (1km)
- Lakeside School (3.2km)
- Waukazoo School (4.1km)
- Lakewood School (4.1km)
- Montello Park School (4.4km)
- School Number 1 (4.8km)
- Westside Christian School (5.2km)
- Christian High School (5.2km)
- Knoll School (5.7km)
- Van Raalte School (5.8km)
- Jefferson School (5.9km)
- Saint Francis de Sales School (6.4km)
- Washington School (6.5km)
- Woodside School (6.6km)
- Beechwood School (6.6km)
- Maplewood Christian School (6.7km)
- Christian Junior High School (6.8km)
- South Side Christian School (7km)
Local Feature A Nearby feature worthy of being marked on a map..
- South Shore Marina (0km)
- Middle Ground Light (0.6km)
- Macatawa Bay Yacht Club (2km)
- Bayshore Yacht Club (2km)
- Bayshore Marina (2.2km)
- Anchorage Marina (2.3km)
- Parkside Marina (2.4km)
- South Pierh Light (3.2km)
- North Breakwater Light (3.6km)
- Green Mountain Beach (6.4km)
populated place a city, town, village, or other agglomeration of buildings where people live and work.
- Virginia Park (0.5km)
- Macatawa (2.7km)
- Tasmas Corners (4.4km)
- Castle Park (4.5km)
- Graafschap (5km)
- Beechwood (5.9km)
- Holland (7km)
park an area, often of forested land, maintained as a place of beauty, or for recreation.
- Jenison Park (1.5km)
- Central Park (2km)
- Holland State Park (2.5km)
- Macatawa Park (2.8km)
- Kollen Park (5.7km)
Accommodation around South Shore Marina
CityFlats Hotel 61 East 7th street, Holland
Comfort Inn Holland 422 E 32nd St, Holland
Days Inn Holland 717 Hastings Ave, Holland
bay a coastal indentation between two capes or headlands, larger than a cove but smaller than a gulf.
- Big Bay (1.5km)
- Macatawa Bay (2.1km)
- Pine Creek Bay (3.5km)
lake a large inland body of standing water.
- Macatawa, Lake (1.5km)
- Kelly Lake (3.9km)
- Gilligan Lake (6km)
stream a body of running water moving to a lower level in a channel on land.
- Kelly Lake Drain (3.8km)
- Pine Creek (4.4km)
- Macatawa River (5.6km)
cemetery a burial place or ground.
- Graafschap Cemetery (4.2km)
- Lakewood Cemetery (4.4km)
post office a public building in which mail is received, sorted and distributed.
church a building for public Christian worship.
- Harderwyk Church (3.8km)
- Fellowship Church (4.5km)
beach a shore zone of coarse unconsolidated sediment that extends from the low-water line to the highest reach of storm waves.
- Ottawa Beach (3.1km)
- Tunnel Park (4.2km)
airport a place where aircraft regularly land and take off, with runways, navigational aids, and major facilities for the commercial handling of passengers and cargo.
- Park Township Airport (3.2km)
administrative division an administrative division of a country, undifferentiated as to administrative level.
- Park, Township of (4.3km)
mountain an elevation standing high above the surrounding area with small summit area, steep slopes and local relief of 300m or more.
- Baldy (4.8km)
cape a land area, more prominent than a point, projecting into the sea and marking a notable change in coastal direction.
- Superior Point (2.4km)
Wikipedia entries close to South Shore Marina
- Holland Harbor Light tower landmark
- Holland (Michigan) city h city
- Tulip City Airport airport airport
- Heath Township, Michigan city o
- Agnew, Michigan city o
- Salem Township, Allegan County, Michigan city o
- Grand Haven, Michigan city o
- Eastmanville, Michigan city o
- Pullman, Michigan city o
- Spring Lake, Michigan city o city
- Grand Valley, Michigan city o
- Tallmadge Township, Michigan city o
- Dorr Township, Michigan city o
- Padgham Field airport airport
- Marne, Michigan city o
- Black River (Southwest Michigan) river
Airports close to South Shore MarinaGerald r ford international(GRR), Grand rapids, Usa (65km)
Capital city(LAN), Lansing, Usa (154.2km)
General mitchell international(MKE), Milwaukee, Usa (167.5km)
Waukegan rgnl(UGN), Chicago, Usa (170.7km)
Chicago ohare international(ORD), Chicago, Usa (198.7km)


text:
About html programming
As with the resume, I would counsel putting HTML and XML below a section like 'Systems'. I commonly have a section like this exactly where I checklist such things as Variation Manage software, OS's I have formulated for, Construct units, etcetera.
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Initially designed for incorporating modest amounts of interactivity to a web page (like hovers and animations - you realize the kind of issue), JavaScript has become made use of for almost everything approximately massive apps and video games, and can even be located in servers.
Web environments are dynamic, factors materialize continuously: the web page hundreds from the browser, the user clicks a button or moves the mouse over a backlink, etc. These are typically referred to as events (which will be the matter of lesson 3).
The lang attribute identifies the natural language in the component's contents, which may be distinctive from that of the rest of the document. By way of example, within an English-language doc:
The element possibly is made up of scripting statements, or it points to an exterior script file from the src attribute.
AJAX (Asynchronous JavaScript and XML) is definitely the buzzword of 21st century World-wide-web applications, and JavaScript is an enormous participant. Within this lesson you learn how to generate your Web content behave smoothly and responsively the same as a desktop application.
This training course focuses on creating multiplatform mobile applications utilizing the Website systems (HTML5, CSS and Javascript). Especially we take advantage of the Cordova hybrid application framework to produce and goal various cellular System
When you are observing this information, it means we are possessing hassle loading external methods on our website.
And sod's regulation dictates the a single you do miss out on will be the one that might have saved you times of work on your latest job...
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text:
Mumbai, Aug 7 (IANS) The Indian equity markets on Monday traded rangebound and closed on a flat-to-negative note on the back of outflow of foreign funds and heavy selling pressure in IT and Teck (technology, media and entertainment) stocks.
The wider Nifty50 of the National Stock Exchange (NSE) fell by 9 points or 0.09 per cent at close at 10,057.40 points.
The 30-scrip Sensitive Index (Sensex) of the BSE, which opened at 32,377.80 points, closed at 32,273.67 points — down 51.74 points, or 0.16 per cent — from its previous close at 32,325.41 points.
The Sensex touched a high of 32,396.14 points and a low of 32,235.82 points during intra-day trade.
However, the BSE market breadth was bullish with 1,550 advances and 1,055 declines.
In terms of the broader markets, the S&P BSE mid-cap index rose by 1.06 per cent and the small-cap index by 1.15 per cent. The mid-cap index touched a fresh high of 15,631.85 points during the day's trade.
"Markets ended marginally lower on Monday after a rangebound session. The main indices stayed in a small range of trade," Deepak Jasani, Head of Retail Research, HDFC Securities, told IANS.
"Major Asian markets have ended on a mixed note. European indices like FTSE 100 and CAC 40 traded higher," said Jasani.
On the currency front, the rupee weakened by 21-22 paise to close at 63.80 to a US dollar from its previous close at 63.58-59.
In investments, provisional data with the exchanges showed that foreign institutional investors (FIIs) sold scrips worth Rs 199.21 crore, while domestic institutional investors (DIIs) purchased stocks worth Rs 308.15 crore.
"Indian shares edged higher on the first trading day of fresh week, as Tata Steel rose on expectations of strong quarterly results. However, broader gains were capped by profit-taking on concerns over valuations," Dhruv Desai, Director and Chief Operating Officer of Tradebulls, told IANS.
"Both the NSE index and BSE index hit record highs last week, raising concerns that factors such as positive earnings and hopes of an improving economy were already factored into prices. Top gainers on the NSE were Tata Steel, Indian Oil Corporation and State Bank of India (SBI), while on the losing front were Infratel, NTPC and Infyosys," Desai added.
Sector-wise, the S&P BSE IT index fell by 97.42 points, the Teck index by 48.88 points, and the telecom index was a tad lower by 2.05 points.
On the other hand, the S&P BSE oil and gas index surged by 237.28 points, the metal index by 145.41 points and the consumer durables index by 142.11 points.
Major Sensex gainers on Monday were: Tata Steel, up 4.26 per cent at Rs 600; SBI, up 1.85 per cent at Rs 311.10; Adani Ports, up 1.59 per cent at Rs 413.35; ICICI Bank, up 1.28 per cent at Rs 299.95; and Coal India, up 0.84 per cent at Rs 251.30.
Major Sensex losers were: Infosys, down 1.75 per cent at Rs 968.15; Tata Motors (DVR), down 1.72 per cent at Rs 251.15; NTPC, down 1.64 per cent at Rs 174.35; Dr. Reddy's Lab, down 1.51 per cent at Rs 2,210.30; and Mahindra and Mahindra, down 1.34 per cent at Rs 1,399.30.
Post Source: Ians feed


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PHP developer jobs in gurgaonPHP Developer/2-5 years/Gurgaon/5 days working/91-9999125805/ firstname.lastname@example.com
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Company Name: MY Taxi India Pvt Ltd
Designation: PHP Developer
Location: Gurgaon
Experience: 2-5 years
Salary : Best in industry(Hike on current salary)
1. We are looking for a good PHP developer with 2+ years of experience .
2. Good knowledge of Core PHP concepts. Should have knowledge of any one framework like Laravel, CodeIgniter, CakePhp, YII
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6. Requirement gathering and analysis skills.
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Key Skills: - Core PHP, MY SQL, Java script, J-query, HTML, Code Igniter, Cake PHP, CSS
UG: B.Tech/B.E. - Computers, Diploma - Computers, B.Sc - Computers, BCA - Computers
PG: M.Tech (Computers), MCA - Computers, M.Sc Computers, PG Diploma - Computers


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Since Shuddle imploded mid-April, a lot of those rides for unaccompanied minors are going to Zūm, a San Mateo startup. Zum needs drivers all over...
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text:
|Pharmaceutical compositions and methods for treating cancer and biomarkers for drug screening
|Echeverria Moran et al.
This application is a divisional of co-pending application Ser. No. 14/617,318, filed Feb. 9, 2015, which is a Divisional of application Ser. No. 13/903,504, filed on May 28, 2013, now abandoned, for which priority is claimed under 35 U.S.C. §120, the entire contents of all of which are hereby incorporated by reference.
1. Field of the Invention
The present invention relates to a pharmaceutical composition for treating cancer, comprising sorafenib and GW5074. The invention also discloses a method for dissociating a protein complex consisting of c-Raf and DAPK by drugs, which can then be used as targets for designing new drugs. Additionally, the invention further provides a method for screening drugs utilizing proteins c-Raf and DAPK as well as the phosphorylation status of DAPK.
2. Description of the Prior Art
Activation of the proto-oncogenes or deficiency of the tumor suppressor genes often leads to cancer cell development. Ras is a proto-oncogene and activation of Ras protein is normally triggered by receptor tyrosine kinase (TKIs) on the cell membrane. Activated Ras protein binds with RAF and subsequently transmits the signal downstream to activate the MAPK pathway which, in turn, regulates cell growth as well as cell differentiation. Activated Grb-sos protein arising from binding of a growth factor to its receptor on the cell membrane leads to phosphorylation of downstream Ras-GDP protein, and the resulting Ras-GTP then binds to the N-terminus of Raf protein and activates Raf, which further regulates the activation of ERK through phosphorylation of MEK. Next, activated ERK enters the cell nucleus and induces cancer cell proliferation. Therefore, countless drugs that are designed specifically against these proto-oncogenes such as these tyrosine kinase inhibitors (TKIs) were generated, only to discover that many cancer patients developed drug resistance during the treatment. Consequently, combination therapy that specifically targets the signaling transduction pathway of tyrosine kinase has become a common treatment method.
Angiogenesis as well as cell proliferation play essential roles in tumor growth. Binding of vascular endothelial growth factor (VEGF-A) released from cancer cells in vast amounts to vascular endothelial growth factor receptor (VEGFR-2) on the surface of the endothelial cells of a tumor activates the signaling transduction pathway of Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) which in turn induces angiogenesis of endothelial cells. Meanwhile, the Ras-ERK pathway also facilitates cancer cell proliferation. In addition, loss of regulation of the Ras-Raf-ERK pathway has been shown in a number of tumor cell lines. Thus, VEGF and Raf may be the best targets for inhibition of tumor growth.
Sorafenib (Nexavar®, BAY 43-9006, Bayer HealthCare Pharmaceuticals) is an oral Multi-Kinase Inhibitor commonly used for treating various cancers. Sorafenib can inhibit proteins such as Raf, VEGF receptor, platelet-derived growth factor (PDGF) receptor, KIT and Fms-like tyrosine kinase-3 (FLT-3). A number of studies have indicated that sorafenib inhibits tumor growth by inhibiting Raf signaling pathway in different cancer cells while suppressing proliferation of the endothelia cells surrounding cancer cells through inhibition of VEGF as well as PDGF signaling pathways, and subsequently induces cancer cell death. The design and test results of these drugs are ideal. However, after few years of clinical applications, it was unfortunate discovered that, although the tumor size at the early stage of treatment was efficiently inhibited by sorafenib, the drug not only cannot eradicate the tumor completely, but causes serious side effects. Moreover, these treated cancer cells developed drug resistance after prolonged treatment. Additionally, in recent years, certain studies have shown that inhibition of Raf signaling pathway in cancer cells leads to reduced inhibition of sorafenib due to alternative regulation by different molecules in cancer cells. Most importantly, some evidence further indicated that the inhibition effects of sorafenib on cancer cells may not be regulated by the Raf pathway. Hence, the aforementioned imperfections need to be further improved.
Treating cancer patients with sorafenib at an early stage can indeed efficiently inhibit tumor size. Nonetheless, this treatment cannot eliminate tumors completely. Even worse, severe side effects usually appear after prolonged treatment and tumor cells may consequently develop drug resistance. This indicates that the current treatment is not an effective method and further improvements are urgently required. Therefore, the present invention reveals certain discoveries, comprising: (1) a novel combination therapy of sorafenib and GW5074; (2) a target for new drug design to effectively kill cells by triggering cell necroptosis induced by dissociation of c-Raf and DAPK; and (3) a biomarker used for drug screening utilizing proteins c-Raf and DAPK as well as the phosphorylation status of DAPK.
In one aspect, the present invention provides a pharmaceutical composition for treating cancer, comprising sorafenib and GW5074 (3-(3,5-Dibromo-4-hydroxy-benzylidene)-5-iodo-1,3-dihydro-indol-2-one).
According to the invention, the composition is administered separately, concurrently, or orderly.
According to the invention, the cancer comprises renal cell carcinoma, prostate cancer, breast cancer, lung cancer, cervical carcinoma, oral cancer, glioma, urothelial cell carcinoma, or melanoma.
According to the invention, the pharmaceutical composition further includes pharmaceutically acceptable salts or vehicles. The aforementioned vehicles comprise excipients, diluents, thickeners, fillers, binders, disintegrants, lubricants, oil or non-oil agents, surfactants, suspending agents, gelling agents, adjuvants, preservatives, antioxidants, stabilizers, coloring agents or spices thereof.
According to the invention, the pharmaceutical composition is for oral administration, immersion, injection, topical application or patch administration.
According to the pharmaceutical composition of the invention, GW5074 binds to c-Raf protein (SEQ ID NO: 1) and induces conformational change of c-Raf, which consequently increases the binding affinity of sorafenib to altered c-Raf protein and disassembles c-Raf from DAPK protein complex.
In another aspect, the present invention reveals the applications of any one of the abovementioned pharmaceutical compositions for treating cancer.
In another aspect, the present invention also provides methods for preparation of drugs that separate a protein complex using compounds, wherein at least one of the compounds can be used for dissociation of a protein complex and said protein complex consists of c-Raf (SEQ ID NO: 1) and DAPK (SEQ ID NO: 2).
According to the invention, the composition comprises sorafenib and GW5074.
In one aspect, the invention discloses a method for drug screening utilizing a biomarker and comprises a provided specimen and at least one of the following: the expression status and/or phosphorylation status of a biomarker prior to administration of the drugs. Drugs that are positively correlated with growth inhibition of cells after administration are deemed suitable drugs. The biomarker is selected from at least one of the following: protein c-Raf (SEQ ID NO: 1) and protein DAPK (SEQ ID NO: 2).
According to the invention, the phosphorylation site of c-Raf protein and DAPK protein is serine 338 (Ser338) and serine 308 (Ser308), respectively.
According to the invention, the specimen comprises ascites, blood, urine, feces, sputum, mucosal cells, gastric fluid, bile, or detached cancer tissues collected after a surgery.
According to the invention, the drugs are sorafenib and GW5074.
In one embodiment, the present invention also discloses the use of a biomarker for drug screening, wherein the biomarker is at least one of the following: protein c-Raf (SEQ ID NO: 1) and protein DAPK (SEQ ID NO: 2).
According to the invention, the phosphorylation site of c-Raf protein and DAPK protein is serine 338 (Ser338) and serine 308 (Ser308), respectively.
According to the invention, the drugs are sorafenib and GW5074.
According to the invention, the use further includes detection of at least one of the following: the expression status and phosphorylation status of the biomarker prior to administration of drugs, and those drugs that are positively correlated with growth inhibition observed after treatment are deemed suitable drugs.
The concept of the novel combination therapy is combining sorafenib, which is originally an antiangiogenic drug, with GW5074, which is a c-Raf inhibitor. When administered singularly at a low-dose (sorafenib at 5 uM or GW5074 at 10 uM), none of the two drugs showed growth inhibition. However, the combination of these two originally non-toxic drugs produced cytotoxicity, considerably reduced side effects resulting from an effective dose of sorafenib, as well as inhibited cancer cell growth. The novel combination therapy revealed in the present invention uncovers a unique molecular mechanism demonstrating that these two drugs destroy the protein complex of c-Raf and DAPK by binding to c-Raf, which in turn leads to cell apoptosis and can be used as new targets for future drug design. Studies indicated that the combination therapy was most effective in cancer cells with highly phosphorylated c-Raf s338, while exhibiting poor inhibition effects on human cancer cells with low DAPK protein expression or lower phosphorylated DAPK-s308.
The drug or drug for screening that dissociates c-Raf and DAPK proteins is a drug combination comprising sorafenib and GW5074.
The term "pharmaceutically acceptable excipient" is used herein to refer to any physiologically inert or pharmacologically inactive substance known to a person skilled in the art that is physically or chemically compatible with either sorafenib or GW5074. Pharmaceutically acceptable excipients include, but are not limited to, polymers, resins, plasticizers, fillers, lubricants, diluents, binders, disintegrants, solvents, cosolvents, surfactants, preservatives, sweeteners, flavoring agents, pharmaceutically grade dyes or pigments, and viscosity agents.
In this application, the term "pharmaceutical composition" is a solid or liquid composition whose form, concentration and degree of purity are suitable for administration to a patient (such as a human or an animal patient) and may induce desired physiological changes after its administration. A pharmaceutical composition is typically sterile and/or non-pyrogenic.
As used herein, the term "combination" describes materials that combining combine two or more compounds and/or drugs (also called ingredients in this application). The term "combined" and "combining" have the same meaning.
Binding of two or more compounds/drugs in a composition can be physical or non-physical. The examples of a compound/drug composition that was bound physically include compositions (e.g. a single mixture) that contains two or more mixed compounds/drugs (e.g. in the same single dosage), a composition that contains two or more chemically/physically-linked compounds/drugs (e.g. by cross-linking, molecular agglomeration or binding to a common vehicle moiety), a composition that contains two or more compounds/drugs that are chemically or physically co-packaged (e.g. formulated in a liquid medium, particles (e.g. micron particles or nanoparticles) or materials on or inside the emulsion droplets, pharmaceutical kits, pharmaceutical packs, or patient packs, wherein two or more compounds/drugs are co-packaged or co-represented (e.g. a batch of dosage units).
The examples of a compound/drug composition with non-physical binding include: a material (e.g. non-single mixture) that contains at least one of the two or more compounds/drugs plus the instructions indicating at least one or more physical bindings that can be used to generate two or more compounds/drugs, a material (e.g. non-single mixture) that contains at least one of the two or more compounds/drugs plus instructions demonstrating a combination therapy utilizing two or more compounds/drugs, a material which contains at least one of the two or more compounds/drugs plus instructions for administration to a patient population, wherein the patient population has been treated with (or is taking) the other drug (others) of the two or more compounds/drugs, and a material that comprises at least one of the two or more compounds/drugs whose amount or form is specially formulated to be used in composition with the other (others) of the two or more compounds/drugs.
In this application, the term "combination therapy" is used herein to refer to therapy using the composition containing two or more compounds/drugs (as defined above). Thus, in the present invention, "combination therapy," "combination," as well as "composition" of compounds/drugs are used to refer to compounds/drugs administered as part of the complete treatment. Consequently, the posology of each of the two or more compounds/drugs may be different, suggesting that each can be administered at the same or at different times. It is important to understand that the compounds/drugs of said composition may be given in order (e.g. before or after) or concurrently (e.g., at the same time), and may be formulated either in the same pharmaceutical mixture (together) or in different mixtures (separately). Furthermore, simultaneous administration in the same mixture is given as a single mixture, and simultaneous administration in different mixtures is not given as a single mixture. In addition, the posology of each of the two or more compounds/drugs in a combination therapy may also vary in accordance with ways of administration.
These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying Drawings.
FIG. 1a shows growth inhibition of ACHN cells at 24, 48 and 72 hours after single-dose treatment of c-Raf inhibitors (sorafenib, GW5074, L779450 and PLX4720).
FIG. 1b shows growth inhibition of ACHN cells at 24, 48 and 72 hours after combination therapy of various c-Raf inhibitors (Sor: sorafenib, GW: GW5074, L77: L779450, PLX: PLX4720).
FIG. 2 shows the xenografted tumor volumes measured every 3 days for up to 21 days following sorafenib and GW5074 treatments administered singularly or in combination.
FIG. 3 shows the photon signals collected from the immnunodeficient mice orthotopic xenografted with Luc-ACHN-LL cells following treatments of various drugs. To calculate the number of tumors distributed throughout the body, the total photons emitted from the entire body of each mouse was measured as well as quantified by Xenogen® living image software. The arrow indicates the time of initial drug administration.
FIG. 4a shows the status of apoptosis or necrosis of ACHN cells at 24 hours following treatments of DMSO (control group), 5 μM sorafenib and 10 μM GW5074 or combination therapy.
FIG. 4b shows the phosphorylation status of pDAPKS308 and DAPK protein expression of ACHN cells at 24 hours following treatments of DMSO (control group), 5 μM sorafenib and 10 μM GW5074 or combination therapy by immunoblotting.
FIG. 4c shows the immunoprecipitation results of endogenous DAPK (upper) and c-Raf (lower) of ACHN cells at 24 hours after treatments of DMSO (control group), 5 μM sorafenib and 10 μM GW5074 or combination therapy, followed by immunoblotting to stain other relevant proteins using specific antibodies.
FIG. 4d shows growth inhibition of ACHN cells following combination therapy in the presence or absence of PP2A inhibitor: cantharidin (C.A.) or okadaic acid (O.A.).
FIG. 4e shows growth inhibition of ACHN cells stably expressing wild-type or c-Raf mutants at 24 hours following combination therapy. (*p<0.05, **p<0.01 in comparison with wild type c-Raf).
FIG. 4f is a protein expression profile of ACHN cells expressing flag-tagged-c-Raf or indicated mutants at 24 hours following DMSO (Ctrl.) treatment or combination therapy. Flag-c-Raf was immunoprecipitated from the cell lysates, followed by immunoblotting for indicated antibodies.
FIG. 5a shows growth inhibition of ACHN, 786-O and Rcc-Sut-002 cells transfected with scrambled siRNA (Scr), DAPK siRNA1 (siDAPK-a), or DAPK siRNA2 (siDAPK-b) following combination therapy (upper panel). DAPK and □-catenin expression of the cell lysates were examined by immunoblotting (lower panel).
FIG. 5b shows growth inhibition of HeLa (left panel), MDA-MB231 cells expressing V5-tagged-DAP (right panel) and indicated mutants at 24 hours following combination therapy.
FIG. 5c shows growth inhibition of various cancer cells or normal cells at 24 hours following treatments of 5 μM sorafenib, 10 μM GW5074, or combination therapy. The lowest part indicates the phosphorylation status of pDAPKS308 of ACHN group in comparison with the control group. The expression of pDAPKS308 and DAPK was detected by immunoblotting.
FIG. 5d is a regression plot demonstrating the correlation between the expression of pDAPKS308 and growth inhibition of cells. R2=0.4551, R=0.6746, p=0.00001.
FIG. 6a shows the expression status of pDAPKS308 and DAPK in tumors (pT) or normal tissues (pN) (upper) of the same patient by immunoblotting. The lower panel indicates the relative intensity of pDAPKS308 after normalized to GAPDH expression.
FIG. 6b is the immnunohistochemical (IHC) analysis of pDAPKS308 expression of human normal and renal carcinoma tissues. Original magnification, ×40 (upper panel), ×100 (lower panel), scale bars is 100 □M
FIG. 6c shows pDAPKS308 expression of human normal and renal cancer tissues after quantification using microarrays. The upper indicates normal as well as different grades (G), and the lower shows different stages (T) as well as metastasis of cancer cells (meta.).
FIG. 6d is a computer simulation photo demonstrating the conformation of c-Raf kinase after interaction with sorafenib and GW5074. Green represents GW5074 and magenta represents sorafenib.
The present invention will now be described more specifically with reference to the following embodiments, which are provided for the purpose of demonstration rather than limitation.
Human renal carcinoma (ACHN) cells were cultured in Eagle MEM (Minimum Essential Medium) with 10% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin. Sulforhodamine B (SRB) is a negative protein with a sulfonic acid group and binds to basic amino acids of intracellular proteins in weak acidic conditions. The SRB protein was extracted from cells using weak alkaline solution and then subjected to absorbance measurement. The amount of intracellular proteins, which is an indicator for cell survival, can be calculated from the amount of SRB. ACHN or A498 cells were treated singularly with 2.5 μM sorafenib, 5 μM sorafenib, 10 μM sorafenib, GW5074, L779450 or PLX4720 for 24, 48 and 72 hours, followed by SRB assay to assess cell survival. Alternatively, ACHN cells were pre-treated with 10 μM GW5074, 10 μM L779450 or 10 μM PLX4720 for 30 minutes prior to 5 μM sorafenib treatment for additional 24, 48 and 72 hours, followed by SRB assay to assess growth inhibition.
Based on the results, combination of c-Raf inhibitors comprising sorafenib and GW5074 showed no growth inhibition of cells following single low-dose treatment for 24 hours (5 μM sorafenib and 10 μM GW5074) (FIG. 1a, mean±S.D., n=4). However, combination of these two drugs not only induced cytotoxicity which was not observed in separate treatment of either drug originally, but successfully reduced side effects caused by effective dosage of sorafenib and inhibited cancer cell growth, suggesting a synergistic effect (FIG. 1b, mean±S.D., **P<0.01, n=4).
Six-week old immnunodeficient male mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl) with an average weight of 20 grams were xenografted with 1×107 ACHN cells to the right by intraperitoneal injection (i.p.). The mice were maintained in a specific pathogen-free (SPF) environment. The size of tumor was measured using a digital caliper twice a week and tumor volume was calculated by the equation of length*width*height*0.5. Drug administration which included 5 mg/kg sorafenib by oral gavage and subcutaneous injection of 10 mg/kg GW5074 once a day for three weeks was initiated when the tumor volume was >100 mm3, and the size of tumor was measured every three days. A total of four groups were included in the experiment: a control group which received only vehicle (DMSO), and test groups administered with 5 mg/kg sorafenib, 25 mg/kg GW5074, or combination therapy of 5 mg/kg sorafenib and 25 mg/kg GW5074, respectively, with 8 mice in each group.
The results are shown in FIG. 2. Separate treatment of either 5 μM sorafenib or 10 μM GW5074 exhibited nearly no inhibition effect. In contrast, combination therapy of 5 μM sorafenib and 10 μM GW5074 demonstrated significant growth inhibition of ACHN cells (t-test, mean±SD, **P<0.01, n=8).
To simulate clinical phenomenon, we established an orthotopic spontaneous animal model for studying metastasis of renal carcinoma. First, ACHN cells were transfected with luciferase gene and the resulted Luc-ACHN transfectants (stable transfectants which express luciferase) with different expression status were then selected for in vivo culture in mice by subcutaneous injection. Six-week old and shaved mice were subcutaneously injected with 1×107 Luc-ACHN cells in 0.1 ml PBS. Two months after injection, the mice were sacrificed and their kidneys, livers, regional lymph nodes as well as other organs were collected for assessing tumor cells that were potentially metastatic, which was confirmed by hematoxylin-eosin (H&E) staining. Tumor cell lines that are highly metastatic were dissected in 1 ml PBS into small pieces aseptically and then cultured in MEM media containing 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin following centrifugation. A few days later, monoclonal cell lines were first treated with trypsin to dissociate the cells and then cultured in vitro. The cells obtained from liver tumor tissues were named ACHN-L. The metastatic potential of various monoclonal cell lines collected from different organs was further analyzed by xenografting these tumor cells to the left kidneys of mice. The xenografted mice were sacrificed and examined 8 weeks after injection of tumor cells. Tumor growth observed in each organ was investigated through visual examination and histology procedures. The metastatic cells found in the liver were dissected into small pieces aseptically and cultured in vitro. The cells obtained from liver tumor tissues were named ACHN-L (subcutaneous injection) cells and the cells collected from the metastatic lesions after injection of ACHN-L cells to the left kidneys were called ACHN-LL cells. The same procedure using orthotopic xenograft of ACHN-LL cells and liver metastatic cells was repeated twice to select for highly metastatic tumors. The cancer cell line (Luc-ACHN-LL), which is highly metastatic and causes high mortality, was selected. Immunodeficient male mice was injected with 3×105 Luc-ACHN-LL cells in 50 μL PBS at the right renal capsule. In vivo luciferase activity was examined every day for up to three weeks so as to assure no leakage of cancer cells at the kidneys immediately after injection and to monitor metastasis of these cancer cells. Two to five minutes before utilizing the IVIS Xenogene system, 75 mg/kg D-Luciferin (Xenogen) in PBS was injected to the retro-orbital sinus of each mouse. Two weeks after injection of Luc-ACHN-LL cells, IVIS Xenogene system was used to monitor transfer of biological fluorescent images and to calculate the intensities of the photon signals (photons/s/cm2/steradian). A total of five groups were included in the study: a control group which received only vehicle, and four test groups which respectively received 10 mg/kg sorafenib, 25 mg/kg GW5074, a combination therapy of 10 mg/kg sorafenib and 25 mg/kg GW5074 (the animals were fed with Sor 30 min post i.p. injection of GW), or 60 mg/kg sorafenib.
As shown in the results, the mice of the control group, 10 mg/kg sorafenib group, and 25 mg/kg GW5074 group died within 5, 8, and 5 weeks following injection of cancer cells, respectively. Likewise, loss of weight was also observed in the high-dose test group which received 60 mg/kg sorafenib, followed by death within 10 weeks. Surprisingly, only mice in the group which received low-dose combination therapy of 10 mg/kg sorafenib and 25 mg/kg GW5074 showed inhibition of both tumor sizes and metastasis of tumors as well as prolonged survival period in comparison with other groups. FIG. 3c shows the IVIS images and quantification of photon intensity, mean±SD, **P<0.01, n=4. In addition, the body weights as well as the energy of the mice that received combination therapy were similar to those observed in normal mice. The tumor cells collected from the mice which received combination therapy also showed profound cell necroptosis in comparison with other groups.
In summary, combination therapy of low-dose sorafenib and GW5074 is effective in growth inhibition of cancer cells in vitro. Also, most importantly, the efficacy of combination therapy can be examined in vivo in the condition which simulates clinical metastasis of cancer cells.
Combination therapy of sorafenib and GW5074 induces cell necroptosis through dephosphorylation of the death-associated DAPK at serine 308 followed by dissociation from proto-oncogene c-Raf. Cells were cultured as described in example 1, washed with phosphate buffered saline (PBS), and stained with annexin V-FITC and propidium iodide (PI) for 15 minutes. The presence of fluorescent Annexin V as well as propidium iodide (PI) was detected by flow cytometry so as to determine whether the cell death is caused by apoptosis or necrosis. The expression of c-Raf and DAPK as well as the phosphorylation status of DAPK were examined by Western blot and immunoprecipitation using antibodies. Anti-DAPK antibodies or non-immune rabbit IgG (IP: immunoglobulin) were included as negative controls, and cell survival was measured by SRB assay, as shown in example 1. In this experiment, ACHN cells were treated separately with DMSO (as a control group), 5 μM sorafenib, 10 μM GW5074, or combination therapy of 5 μM sorafenib and 10 μM GW5074 for 24 hours.
The results indicated that the average cell necroptosis rate of ACHN cells treated with DMSO, monotherapy of 5 μM sorafenib, and monotherapy of 10 μM GW5074 was 0.37, 0.77, and 1.35%, respectively. However, cells which received combination therapy showed significant increase of necroptosis, up to 53.95% (FIG. 4a, t-test, mean±SD, ***P<0.001, n=4). Significant decrease in phosphate groups of S308 due to dephosphorylation of pDAPKS308 by PP2A in tumor cells was only observed in cells treated with combination therapy (FIG. 4b). Results from immunoprecipitation further suggested c-Raf and DAPK, as well as PP2A, formed a complex. Combination therapy reduced the interactions among PP2A, DAPK and c-Raf, which subsequently decreased pDAPKS308 (FIG. 4c). For the test group which received combination therapy, various concentrations of PP2A inhibitors (Cantharidin acid, C.A. and Okadaic acid, O.A.) were effective in inhibition of dephosphorylation of pDAPKS308 and growth of ACHN cells only when provided 30 minutes prior to administration of the combination drugs (FIG. 4d, *P<0.05, **P<0.01, n=4).
Next, the effects of combination therapy on the efficacy of pc-RafS338. Reduction of pDAPKS308 noted as being most significant when ACHN cells were transfected with c-RafS338D (simulation of phosphorylating S338). Moreover, c-RafS338A (simulation of dephosphorylating S338) not only weakened dephosphorylation of DAPK S308, but also reduced growth inhibition of ACHN cells under combination therapy (FIG. 4e, *P<0.05, **P<0.01, n=3). In addition, immunoprecipitation results suggested that c-RafS338D (simulation of phosphorylating S338) apparently lost its interaction with PP2A in comparison with c-RafWT (wilt type) and c-RafS338A (simulation of dephosphorylating S338) (FIG. 4f). Therefore, combination therapy not only increases pc-RafS338, but facilitates dephosphorylation of pDAPKS308 by PP2A and enhances cell necroptosis.
The above-mentioned example reveals that combination therapy of sorafenib and GW5074 induces cell necroptosis through dephosphorylating serine 308 of death-associated protein kinase (DAPK) followed by dissociation from proto-oncogene c-Raf. Disassembly of c-Raf and DAPK can also be used as the target for future drug design, which is dissociation of c-Raf from DAPK in cells leads to initiation of cell necroptosis process which kills cells effectively.
Previous studies showed that dephosphorylation of DAPK at serine 308 can be used as a predictive biomarker for anticancer effects of drugs on cancer cells. The methods for cell culture as well as detection for examining growth inhibition are as described in example 1. ACHN, 786-O and Rcc-Sut-002 (drug-resistant cancer cells) cell lines were treated with siRNA of type a and type b DAPK (siDAPK-a, sense strand: 5′-CAAGAAACGUUAGCAAAUGUU-3′ [SEQ ID No:3] and antisense strand: 5′-CAUUUGCUAACGUUUCUUGUU-3′ [SEQ ID No:4]; siDAPK-b, sense strand: 5′-GGUCAAGGAUCCAAAGAAGUU-3′ [SEQ ID No:5] and antisense strand 5′-CUUCUUUGGAUCCUUGACCUU-3′ [SEQ ID No:6]). Each cell line was analyzed by siDAPK-a, siDAPK-b and a control (Scr). Growth inhibition of cells was examined following combination therapy of 5 μM sorafenib and 10 μM GW5074 for 24 hr. On the other hand, HeLa cells and MDA-MB-231 cells were transfected with the control empty vector (vector), WT (wild type), DAPKS308D, DAPKS308A or DAPKK42A (inactivated protein kinase), and growth inhibition of the transfected cells was examined following combination therapy of 5 μM sorafenib and 10 μM GW5074 for 24 hours.
The results indicated that DAPK protein is indispensible for cytotoxicity induced by combination therapy of sorafenib and GW5074. In ACHN, 786-O and RCC-SUT-002 cells, growth inhibition resulting from two siRNAs capable of inhibiting DAPK expression was reduced by around 60% (FIG. 5a). Furthermore, phosphorylation of DAPK at S308 is highly associated with cytotoxicity induced by combination therapy. Overexpression of DAPKWT and DAPKS308D in HeLa cells which usually express higher pDAPKS308 notably increased the cytotoxicity after receiving combination therapy, whereas cell death was not enhanced in DAPKK42A (inactivated DAP kinase) and DAPKS308A (simulation of non-phosphorylation of S308) following combination therapy. Only DAPKS308D was found to increase growth inhibition in MDA-MB-231 breast cancer cells which usually express lower pDAPKS308 after receiving combination therapy. The rest, including DAPKWT, DAPKS308A, and DAPKK42A, showed no increase in growth inhibition (FIG. 5b, mean±SD, **p<0.01, n=3). According to the results, it is not the DAPK protein itself, but S308 phosphorylation of DAPK which plays a key role in inducing cytotoxicity when treated with combination therapy of sorafenib and GW5074. This is because dephosphorylation of S308 is not effective under combination therapy even with higher DAPK expression. The cytotoxicity effect induced by combination therapy must go through activated DAP K. Therefore combination therapy of sorafenib and GW5074 is only effective in the presence of pDAPKS308 in cancer cells.
Phosphorylation of c-Raf protein can be used as a biomarker for predicting the anticancer effect of drugs on cancer cells. Reduction of pDAPKS308 is most evident in ACHN cells transfected with c-RafS338D (simulating phosphorylation of S388). Not only does c-RafS338A (simulating dephosphorylation of S388) reduce dephosphorylation of DAPK S308, but also decreases growth inhibition of ACHN cells under combination therapy (FIG. 4e, *P<0.05, **P<0.01, n=3). Thus, S338 phosphorylation of c-Raf is beneficial for combination therapy due to better treatment effects, and c-Raf S338D (simulating phosphorylation of S388) has a better effect in comparison with c-Raf S338A (simulating dephosphorylation of S388).
In addition, a number of cancer cells as well as normal cells were examined. It was found that combination therapy caused only limited cytotoxicity in the above-mentioned cells due to low S308 phosphorylation of DAPK in normal fibroblasts and epithelial cells. Nonetheless, growth inhibition caused by combination therapy among various tumor cells is positively correlated with the status of S308 phosphorylation of DAPK (FIGS. 5c and 5d, mean±SD, **p<0.01, n=3). It was further investigated as to whether combination therapy has an inhibition effect on drug-resistant cancer cells using sorafenib-resistant cancer cells obtained from clinical cases (RCC-Sut-001, RCC-Sut-002, RCC-Sor-001) and animal models (786-OT4, ACHN-T2R). All drug-resistant cancer cells that have highly phosphorylated DAPK S308 were significantly inhibited under combination therapy. Moreover, HT29 and A2058 cancer cell lines that are both Raf inhibitors-resistant showed high S308 phosphorylation of DAPK as well, and combination therapy demonstrated significant inhibition as well as synergistic effect (FIG. 5c and Table 1). Additionally, because S308 phosphorylation of DAPK in normal cells are relatively low, growth inhibition of cancer cells induced by combination therapy of sorafenib and GW5074 are therefore selective and cause no toxicity in normal cells.
|renal cell adenocarcinoma
|renal cell adenocarcinoma
|renal cell adenocarcinoma
|prostate carcinoma
|prostate carcinoma
|prostate carcinoma
|prostate carcinoma
|breast adenocarcinoma
|breast adenocarcinoma
|breast adenocarcinoma
|lung carcinoma
|non-small cell lung cancer
|hepatocellular carcinoma
|cervix carcinoma
|submaxillary salivary gland carcinoma
|glioblastoma; astrocytoma
|brain malignant glioma
|kidney epithelial
|embryo fibroblast
|uroepithelium epithelial
|prostate normal epithelial cell
|prostate normal epithelial cell
|colorectal adenocarcinoma
|colorectal adenocarcinoma
|B-Raf mutation
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
|drug-resistant renal cell
|Acquired resistant
|adenocarcinoma cell
c-Raf and DAPK are found in cytoplasm and mitochondria. Combination therapy leads to relocation of DAPK between cytoplasm and mitochondria, along with dephosphorylation of pDAPKS308 by PP2A. Dephosphorylated DAPK decreases its interaction with c-Raf in cytoplasm. In addition, only DAPKS308D can be induced to be translocated from mitochondria to cytoplasm of MDA-MB-231 under combination therapy. This results in production of ROS and low phosphorylation of pDAPKS308. However, reduction of both c-Raf and phosphorylation of its S338 induced by combination therapy in cytoplasm and mitochondria was found only in cancer cells with highly phosphorylated pDAPKS308 and not in cancer cells with low pDAPKS308.
The tumor samples and normal tissue samples collected from 20 patients with renal cell carcinoma were further investigated. Based on the Western blot results, 16 out of 20 samples showed elevated phosphorylation of DAPK at S308 in cancer cells in comparison with normal tissue samples (FIG. 6d. (pDAPKS308/GAPDH, mean±S.D., **p<0.005). Immnunohistochemical (IHC) analysis also demonstrated that S308 phosphorylation of DAPK was higher in 181 human renal carcinoma samples in comparison with normal kidney tissues (FIG. 6b). On the other hand, S308 phosphorylation of DAPK showed no significant differences between different grades (G) or stages (T) of the cancer using tissue microarray (TMA) and semi-quantitative analysis. The results suggested that pDAPKS308 is not only a factor for determining prognosis, but also a predictive biomarker of combination therapy for treating kidney cancers.
According to the results, a computer simulation experiment was then conducted to assess the crystal structures of various c-Raf inhibitors bound with c-Raf. The maximal energy was expected to be produced by binding of inhibitors to the structural region of c-Raf kinase when the combination of GW5074 and sorafenib binds to c-Raf (−182 kcal/mole, Table 2). GW5074 at the front end binds with c-Raf and produces a deeper hydrophobic pocket for binding through Ile355, Val363, Ala373, Leu406, Trp423 and Phe47. Under the circumstances, more regions in the hydrophobic pocket will be occupied by GW5074 and sorafenib (as shown in FIG. 6d).
|Various inhibitors bind to c-Raf separately or in combinations
|+ Sorafenib
|+ sorafenib
|+ sorafenib
The present invention discloses a novel combination therapy for treating cancer by utilizing a composition that comprises sorafenib and GW5074. The combination therapy is not toxic to normal cells due to selective inhibition on cancer cells. Therefore, this therapy is safe and very promising for future applications. The efficacy of the combination therapy was verified by using pDAPKS308 as a predictive biomarker so as to avoid unnecessary treatments. The research of the present invention overcomes the obstacles faced by current studies for cancer therapies and meets the requirements of an ideal model for preclinical treatments.
Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.


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- Recruiter
- Hawksley's Law Limited
- Location
- Camberley, Surrey
- Salary
- National minimum wage
- Posted
- 01 Oct 2018
- Closes
- 01 Nov 2018
- Practice area
- Civil litigation, Conveyancing - commercial, Conveyancing - residential, Family, Planning law, Private client
- Not applicable
- Position
- Paralegal, Trainee and graduate
- Sector
- Private practice
- Hours
- Full time
- Contract type
- Trainee
Role: Paralegal with view to a Training Contract
Salary: National minimum wage
Commencing: Immediately
Location: Camberley, Surrey
About Us:
Hawksley's Law Limited is an established 2 Partner niche practice in Camberley, Surrey providing services for clients throughout the Home Counties and across the whole of the UK.
We have been in practice for almost 20 years and take pride in the specialist, committed and affordable service we offer our varied client base.
Our practice areas include planning law, wills & probate, residential & commercial conveyancing, family and general civil litigation. We also deal with a high portion of High Court litigation and have landmark cases to our credit.
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Applications are to be made by email attaching a covering letter and CV to firstname.lastname@example.com.


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This information focuses on the twentieth and twenty-initial hundreds of years, covering how the extent to which the Bible mentions the topic, if it can be condemned, and whether or not the a variety of passages apply these days, are becoming contentious matters.
I stay up for the day when equality for LGBTQ citizens is automated and non-controversial, and that laws to shield the LGBTQ Neighborhood are pointless. Right until that point, I will stand with my fellow citizens and combat to safeguard:
Setting up your no cost demo of Bible Gateway Furthermore is straightforward. You're previously logged in with all your Bible Gateway account. Another stage is to enter your payment information and facts. Your bank card won't be charged right up until the trial time period is about. You'll be able to terminate whenever throughout the demo interval.
Check out this verse. Marriage exhibits the connection in between Christ as well as church. This verse demonstrates just how much God loves you. Just one glimpse upward and you have the Lord hooked. He desires to be along with you and when you enter into His existence His coronary heart beats speedier.
To unlock this text for your buddies, use any on the social share buttons on our site, or just copy the url under.
Similarly, gays and lesbians never see the Bible as unequivocally accurate mainly because they are forced by its use against them to read through it extra closely and with much less credulity, primary them to notice its myriad contradictions.[131]
USA Right now Community talked with professionals and persons in the gay Neighborhood about just what the Q usually means, why It can be made use of and who's indicating it.
In case you are a Christian I need you to definitely realize that you will be loved. His love in your case just isn't based mostly upon your efficiency.
For even though comets go in really eccentric orbs in all way of positions, blind fate could hardly ever make the many planets go one and the same way in orbs concentric, some inconsiderable irregularities excepted which can have arisen with the mutual actions of comets and planets on one another, and that may be apt to boost, until This method wishes a reformation.[69]
Usually do not touch what on earth is unclean, and I'll acquire you.' 'I might be a Father for you, and you shall be My sons and daughters, says the Lord Almighty.'"
LGBTQ is just one set of initials getting used. There are other letters and combos -- a lot of that some phone it "alphabet soup."
It appears like you could be having challenges participating in this online video. If that's so, remember to attempt restarting your browser.
12 How does Jehovah truly feel about our willing obedience? His Phrase suggests that we therefore make his "heart rejoice." (Proverbs 27:11) Does our obedience seriously carry joy to the center from the Sovereign Lord on the universe? Certainly, it does—and once and for all rationale! Jehovah produced us as free moral brokers.
So in the traditional sense and reference of 'philosophy', real understanding is reserved to God; finite guy can only check here be the lover of information, not himself the just one who possesses know-how. From the indicating with the passage, the lover with the awareness that belongs only towards the understanding God, the philosophos, gets to be the theophilos, the lover of God."[18]
LGBTQ Options
This information focuses on the twentieth and twenty-initial hundreds of years, covering how the extent to which the Bible mentions the topic, if it can be condemned, and whether or not the a variety of passages apply these days, are becoming contentious matters.


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Gulfing Around
Days: 52-55
Kilometres Travelled: 730
Total States Visited: 16
Total Kilometres: 11,313
We continued our exploration of the Florida coastline from the comfort (and discomfort for the rear passengers) of Josie as we soon realised there was not a lot going on in these parts.
Despite witnessing some kind of drug deal at the skatepark where we stopped for lunch, our afternoon was very uneventful and we arrived in Crystal River to discover the main attraction was closed. With poor internet, no manatees to visit and lots of rain, the highlight of our day was dinner at Denny's - and for the boys, being eyeballed by the local supermarket check-out chick.
Coastal Highway 98 gave us beautiful scenery but not a lot else; our next hotel seemed to be in the middle of nowhere, aka Wakulla Beach. However, it did provide a very pleasant setting to swim, drink and play card games to escape the oppressive heat (an oasis if you like). It also gave us our first authentic American bbq experience at a local restaurant ''Hamaknockers" where we ate a ridiculous amount of meat and vegetables drowned in butter to celebrate more than 200 days of travelling for Sack and Sponge. The festivities continued with 1L cans of Fosters beer found at the local 7eleven, a bag of M&Ms and some riotous laughs at our hotel.
Our journey around the gulf continued, punctuated with scenic coastal drives and bridges, plenty of palm trees, towns with cool names but terrible cafes (Apalachicola, we're looking at you) and swims at beaches the temperature of bathwater. Fort Walton Beach offered us some decent tacos and margaritas on the beachfront (enjoyed on more than one occasion), while the balmy tropical evenings invited us to stay out and enjoy the photographic opportunities of the jetty (also home to a friendly pod of dolphins).
With not a lot going on besides gentle warm beaches, tropical air and plenty of indulgent food, we were practically ready to join the rest of the Florida population in their retirement...


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We offer a convenient bus service between Doune and Edinburgh, Glasgow and Stirling. The Stirling buses are timed to meet Edinburgh, Glasgow and Perth trains in order to give you maximum flexibility. Booking info for 2019 coming soon!
Glasgow pick up point: City Chambers, George Street,
Edinburgh pick up point: Waterloo Place
Stirling pick up point: Bus stop outside Stirling Railway Station
Questions and problems
We've tried to cover most queries on this page but if we've missed something out please send us an email here.


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Home > Tyler Perry's House of Payne > Season 6 > Episode 1
Where's the Payne?
Episode Info
A forced retirement for Curtis creates obstacles, but the drama spikes when Miranda goes missing. Elsewhere, Claretha returns sans husband and moves in with Curtis and Ella. Kim Fields directed the episode.
LaVan Davis
as Curtis "Pops" Payne
as Curtis "Pops" Payne
Cassi Davis
as Ella Payne
as Ella Payne
Allen Payne
as CJ Payne
as CJ Payne
Demetria McKinney
as Janine
as Janine
Lance Gross
as Calvin Payne
as Calvin Payne
Larramie 'Doc' Shaw
as Malik Payne
as Malik Payne
China Anne McClain
as Jazmine Payne
as Jazmine Payne
Denise Burse
as Claretha
as Claretha
Keshia Knight Pulliam
as Miranda
as Miranda
Discussion Forum
Discuss Tyler Perry's House of Payne on our TV talk forum!


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ALICE - Alice and Tuloso-Midway were locked in a pitchers' duel Monday evening. But all it took were a couple of Lady Coyotes' miscues and, eventually, the Cherokees' offense won out.
Tuloso-Midway scored six runs in the sixth inning, including four off a missed outfield fly ball, to beat Alice, 7-0.
The Cherokees were led by pitcher Emily Brittain, who threw seven innings of no-hit ball Tuesday. It was the edge Tuloso-Midway needed to overcome the Lady Coyotes.
After scoring a first-inning run on a Christina Villalon RBI, the Cherokees were held at bay, mostly by the pitching of sophomore Zalysa Perez, who routinely worked herself out of jams. In the second, Tuloso-Midway loaded the bases with no outs. Perez then struck out the next three batters she faced, including standout Kirsten Schwirtlich, to get out of the inning.
The Cherokees came back with a strong third inning. With runners in scoring position and one out, centerfielder Ashley Ramirez caught a line drive and then throw out a potential run at home.
Things finally caught up with Alice in the top of the sixth inning.
Leading only 1-0, Ashley Valdez hit a double and scored two. The Cherokees then loaded the bases and scored four runs off a missed ball in leftfield.
"(Tuloso-Midway) is so good and so fast," Alice coach April Atterberry said. "If you give them an inch, they'll take a mile. They scored in just the blink of an eye."
The 12-0 Cherokees have the District 31-4A crown locked up. Alice dropped to 7-5 and needs to win at least one of its next two to clinch its first playoff bid.
The Cherokees hosted Laredo Nixon Tuesday night. They still have a make-up game on the road against Flour Bluff.


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HTC One (M8) and other HTC handsets released this year could get Sense 7 UI as soon as May 2015
According to ROM developer LlabTooFeR - who recently showed us what Lollipop and Sense 6 look like on the One (M8) - Sense 7 UI should first ship with the HTC Hima (aka One M9), which could be released in March. After that, between May and July 2015, Sense 7 should become available to HTC's 2014 line-up of handsets - almost certainly not including entry-level models (like the Desire 310).
At the moment, it's unclear if devices from 2013 (including the One M7) will receive the new version of the UI. We also don't know exactly what Sense 7 will bring, but we do expect to see major design changes to complement Android Lollipop. What do you expect from Sense 7?
source: @LlabTooFeR
14 Comments
1. dome888 (Posts: 85; Member since: 10 Oct 2012)
one reason HTC fails, late updates.
2. CanYouSeeTheLight (Posts: 1122; Member since: 05 Jul 2012)
Lmao the first flagship phone apart from the almost pure Moto X last year to get kitkat was the HTC One.
12. dome888 (Posts: 85; Member since: 10 Oct 2012)
it stopped after that
3. gagakiller (banned) (Posts: 176; Member since: 08 Oct 2014)
Why do people here hate gagakiller ?
4. aditya.k (Posts: 496; Member since: 10 Mar 2013)
Because, everybody loves gaga.
6. Neo_Huang (Posts: 1067; Member since: 06 Dec 2013)
I don't know...
5. 0xFFFF (Posts: 3806; Member since: 16 Apr 2014)
Stop Making Sense
9. gagakiller (banned) (Posts: 176; Member since: 08 Oct 2014)
stopping Sense doesn't make any sense
7. Tracy1988 (banned) (Posts: 208; Member since: 18 Dec 2014)
I have bought 5 phones in the last 2 years: Xperia ZL, Xperia ZR, Nexus 5, LG E 970 and Xperia Z3C but I haven't been happy with any of them. If the HTC One display is centered, I would buy it in a heartbeat.
10. HOOHH13 (Posts: 22; Member since: 25 May 2014)
How was the ZR going on?
8. hesam96 (Posts: 6; Member since: 12 Dec 2014)
im a big fan of Sense UI, its very beautiful and very fast. sense is best ui between TW, LG's ui, and its even better than android lolipop ui
11. HOOHH13 (Posts: 22; Member since: 25 May 2014)
exactly! simply and nice :)
13. Badelhas (Posts: 10; Member since: 29 Sep 2014)
If the leaked photos of the Lollipop update with sense 6 are true I will be very disappointed if they don't adopt Google Material Design...
14. rowdysheeter (Posts: 74; Member since: 10 Sep 2014)
shouldn't it be "as late as May 2015"?


text:
Dating on the chive
Me being young and dumb at the time thought that would be exciting.
So he drives me a pretty good way from my house and we go to an elementary school. Apparently he was a basketball coach and they had a game that night.
So at this point I notice I have no reception on my phone and I really wanted to leave but I thought maybe we were going somewhere fun afterward so I gave up trying to call someone to rescue me.
I sat and watched this game right next to this dude's mom. Finally the game ended and we go to get in the car and his friend from the restaurant gets in the front seat making me sit in the back. Ominous Pumpkin Got set up on a date with this pretty awkward guy, he picked me up in his car which was sort of cool considering we were in high school but oh my god he was a nervous driver.
I am super uncomfortable with this and I ask what's going on. So nervous in fact that he accidentally ran a red light, lost his shit and ducked into a residential area to sit at the end of a cul-de-sac freaking out thinking the cops were coming.
They basically tell me they want to run a train on me. I tried to explain that if a cop had seen him we'd have already been pulled over but dude was too freaked out.
She said she was self-conscious though so she wanted the lights off. She had a GIANT (or at least it seemed giant) pink purse with the face of a pig on it.The rest of the night was spent talking about how shitty high school is and how much pressure he felt to lose his virginity but didn't even like me that much. b8le __________ Matched on tinder with a guy who decided that a great story to tell in detail on our date was about the time he fisted a lady and tore her up so bad inside that she was bleeding profusely and had to go to the hospital to get stitches.He had a girl he actually liked but wanted to be "experienced". After he was done telling the story, he told me he was glad I told him I was super squeamish, because now he wouldn't try to show me pictures of all the blood he had saved on his phone."I've been on and off online dating for the last 7 years and I figured throwing up some new pics was worth a shot and worst case scenario, send a couple smirks to strangers.While I was rebounding from a 5-year relationship, I went on a sort-of date with this weird dude from my community college who kinda stalked me, but was pretty much harmless. It was awful, the ball wasn't inflated properly so every rotation you hit the floor it hurt, it was wet so there was water inside the ball and we had to jump out at the bottom of the hill into a puddle so wet sock galore. Gave us something to talk about though, been together nearly 4 years 🙂 The Charity Of Me __________ Had a good friend at University who disappeared overseas for a year after we graduated.
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The 25-year-old Montana driver who severely injured two passengers when he struck a tree in December 2011 has been given a 10-year suspended sentence in connection with the crash. Officials say the man entered an Alford plea, which allows a defendant to acknowledge that the prosecution has enough evidence to convict.
Driver gets suspended sentence in injurious crash
Billings Settles DUI Crash Case
The city of Billings has agreed to pay a $222,000 settlement to a man in a wrongful death suit. His wife was killed by a drunken driver who was released by authorities hours before she got behind the wheel again.
Four injured in drunk driving crash
A tragic car accident in Helena, Mont., has left four people injured after colliding near the intersection of North Montana Avenue and Sierra Road. One of the victims was transported to the hospital in critical condition after the Feb. 27 crash. Authorities report that the crash happened at about 5:15 p.m., and alcohol is believed to have played a role.
Three dead after crash on Highway 87
Montana roads are still plagued with car accidents this year, even as the winter weather begins to wane. Yet another collision has left three young victims dead on Highway 87 after a vehicle crossed the road's center line. Two people were ejected from the apparently head-on accident.


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News In Mind – June Update
Revealed: Women who suffer depression or anxiety in their teens have a ONE IN THREE chance of having baby blues
For years, perinatal depression has been viewed as a unique mental disorder that comes out of nowhere, rather than something that can be tied to experiences and treated before pregnancy is even contemplated. A 20 year study conducted by the Murdoch Children's Research Institute in Australia has revealed a link between mothers who have had prior mental health problems and developing a form of depression or anxiety either during or after their pregnancy.
Sedentary behavior 'could increase the risk of anxiety'
Watching TV, playing video games, surfing the web: sedentary activities such as these have long been associated with physical health problems such as obesity and heart disease. A new study, however, suggests that they could also be associated with an increased risk of anxiety
Men more likely to talk about suicidal thoughts with loved ones than doctor, Black Dog Institute study finds
A national study has found men are more likely to discuss suicidal thinking with trusted acquaintances like family, friends or partners than health clinicians.
The research by not-for-profit the Black Dog Institute aims to identify ways to prevent male suicides, which account for about 75 per cent of the total Australian figure.
Could depression start in the womb? Children of mothers suffering mental illness in pregnancy are 'three times more likely to develop the condition'
Research shows that adults are up to three times more likely to suffer from the condition if their mother had it while pregnant with them. It is thought that exposure to high levels of stress hormones in the womb has lasting effects on the unborn child's brain. The researchers said that screening all pregnant women for depression – and treating those who need it – could stop the legacy from being passed on.
Research Ties Depression, Binge Eating
New research shows that subset of people who exhibit high levels of impulsivity during depression show proclivity for binge eating. Their latest study, published in the International Journal of Eating Disorders, furthers researcher's conviction that "negative urgency" might be the single largest predictor of who is and isn't prone to binge eating.
Nearly a third of early adulthood depression linked to bullying in teenage years
Being bullied at school is an unpleasant and relatively common experience for many young people, but does it lead to long-lasting harm? A growing number of studies have found that being bullied is associated with an increased risk of developing symptoms of anxiety and depression, self-harm and even suicidal ideation.
FIFO workers record double the rate of depression compared to others, WA study reveals
A WA study has now found his story is commonplace with fly-in fly-out (FIFO) workers suffering from depression at more than twice the rate of the Australian population. The research, carried out by Edith Cowan University, surveyed 629 FIFO workers from 143 different resource companies across the state. The study found 28 per cent showed significant signs of depression.


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text:
- August 9, 1930 - September 6, 2018
- Springfield, Massachusetts
of Neal's Passing
Share This Obituary
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Memories & CandlesPrevious
"Chip, Michael, Kim, Janet, Sheila,sorry to hear about your father. News travels slow up north. You are all in my thoughts and prayers.Tom...Read More »
1 of 7 | Posted by: A friend
"My deepest sympathy to all of you. I always thought your dad was a great guy. Carolann 'DeMars' MacMaster
2 of 7 | Posted by: Carolann Macmaster - Ludlow, MA
"My heartfelt condolences to all the Chapin family... Always a smile on his face, and a welcoming hug and greeting for all he came in contact with! A...Read More »
3 of 7 | Posted by: Laurie Kakley - Wilbraham, MA
"My thoughts and prayers are with you all.
4 of 7 | Posted by: Bruce Olson - Eestlongmeadow, MA
"I am so sorry for your loss. May the God of comfort be with you during your time of mourning. Isaiah 61:1,2 ; Matthew 5:4
5 of 7 | Posted by: A friend
"Dear Chip and Kathy Janet and RussellSo sorry to read of your Dad's passing.My thoughts and prayers are with you and your family. May happy...Read More »
6 of 7 | Posted by: Pamela Rideout - Blandford, MA
"Offering our deepest condolences during this difficult time.
7 of 7 | Posted by: The Staff of Byron Keenan Funeral Home - MA
Sympathy Flowers
Neal was born on August 9, 1930 and passed away on Thursday, September 6, 2018.
Neal was a resident of Springfield, Massachusetts at the time of passing.
He was born in Elmwood, CT and educated in Springfield, a son of the late Henry and Annie (Perkins) Chapin and was a graduate of Springfield Technical High School and Western New England College earning his degree in Business Management.
Neal was a veteran of the Korean War serving with the United States Navy and was the recipient of the Good Conduct Medal and the National Defense Service Medal.
His funeral will begin on Monday morning, September 10th at 8:30 AM from Byron Keenan Funeral Home, followed by a Liturgy of Christian Burial at 10:00 AM at Mary Mother of Hope Church, 840 Page Blvd., with Rites of Committal and Military Honors to conclude at Hillcrest Park Cemetery, 895 Parker Street, all in Springfield.
Contributions in Neal's memory may be directed to a veteran's organization of one's choice.


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Applications are now being accepted from organizations that propose to build or upgrade off-road vehicle (ORV) trails, and promote rider safety, announced Doug Donaldson, Minister of Forests, Lands, Natural Resource Operations and Rural Development of British Columbia.
"Government is committed to maintaining the province's world-class recreational trails. Many of the trails are in remote and rural areas, and this money will benefit all users. Trail improvements will help to create local jobs, and will also enhance tourism opportunities," said Doug Donaldson, Minister of Forests, Lands, Natural Resource Operations and Rural Development.
The ORV Trail Fund, currently valued at almost $590,000, is awarding up to $100,000 this summer to support the construction and maintenance of trails for off-road vehicles, and to promote the safe and responsible use of ORVs.
The ORV Trail Fund, created in 2017, was established as a part of the Off Road Vehicle Actand regulations. The act requires registration of all off-road vehicles within B.C. A portion of the registration fees collected by ICBC are redirected to the ORV Trail Fund. The Recreation Sites and Trails Branch administers the fund and this is the first application intake.
The application deadline is July 16, 2018. Requests for funding from $1,000 to $25,000 will be considered.
Any legal entity in the province, except for individuals, can apply. This includes: First Nations, Local governments (such as municipalities and regional districts), ORV organizations (including those representing quads, dirt bikes, snowmobiles, motorbikes and side-by-sides), Non-profit societies and Businesses.
Submissions will be adjudicated by provincial recreation staff, with input from provincial-level ORV representatives. Successful applicants will be notified after Aug. 24, 2018.
A five-year plan has been developed to fund future trail recreation opportunities. The planned yearly disbursements will be between $100,000 and $200,000 per year, depending on the growth of the fund.


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The use of hot press, as its name suggests, is to use heat to deal with the pressure of the product, and its heating method can be more than one kind of Austria, which specific?
Heating system is the most important process of hot press in working. The hot water is heated by the sensible heat of hot water. The steam heating is heating the hot pressed plate with the latent heat of vaporization of the steam. The comprehensive utilization rate of heat energy in the over heated water heating system is 40%-50%. It is necessary to use high pressure saturated steam to heat the low temperature water when heating the hot water. When the heat is used, it can be reused by cyclic heating. Because the hot water is recycled, its flow rate is slow, but the heat efficiency is relatively high.
Previous: Hot press on the thermal oil requirements


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ive recently bought this, and it seems great. sometimes dialougue isnt so great to the centre speaker. (B&W CC6). but when i was fiddling with the menus i found an option to turn OFF dolby pro logic. i use the cinema EX B sound field as i believe it is by far the best. when pro logic option is turned off the sound sounds MORE surround, dramatic and has a greater quality to it with more weight. why on earth is this?! even when playing CD'S it produces a surround sound which sounds better. my guess is that it uses less power as it dosnt have to give sound to an extra channel. i think this as when its ON the display has a little box with an S in it. is this intended for 6:1 or something? my rears are to my side anyway so i dont need any rear sound. i have yet to try it on a dvd yet, but tv, game and CD all sound better. if anyone has any idea then please help. thanks. any other tipe for my set up will be gratefully recieved also.
DB1070 amp.
2 B&W 601 front speakers.
1 B&W CC6 centre speaker.
2 B&W 3 series rear speakers.
1 yamaha 100W sub.
thanks.