- Invented some 200 years ago, in Italy. Based on the harpsichord, they differed in that a hammer struck the string where’s the harpsichord plucked the string.
- Incidentally the harpsichord was based on the harp.
- That change in how the note was produced resulted in a completely different instrument, which could be played loud or soft, hence its name piano forte. Which means soft loud, in Italian.
- Contrast that with the harpsichord which can only play at one level as the string is either plucked out it isn’t. There’s no in-between.
- Pianos have strings and each string has to be tuned. An intricate arrangement of pegs hold a strings under tension. These steel strings are held under huge force and the purpose is the keep the strings in tune.
- Despite that force, pianos do need regular tuning. A new piano needs to be tuned 3 or 4 times a year. As they get older twice a year will normally suffice.
- Although made with extreme precision, the main material is wood. So the tuning of the instrument is affected by atmospheric conditions. Damp and humidity are the main culprits here, causing the piano to go out of tune.
- The two main types of piano are the grand piano and the upright piano. The trade off is between size versus quality of sound. And although the baby grand piano is a compromise on the full size grand, it’s still rather large and not everyone has room for these.
- Grand pianos are also faster, which means the keys reset when half way up.on an upright the key has to fully return before it can be pressed again.
- Modern electronic pianos arrived in the 80s. These proved very popular and the sound has improved and before richer over the years. But one of the truly amazing qualities of these electronic instruments, is that their sound can be altered and they can reproduce the sound of any of the famous models of grand pianos out there.
In the world of keyboards everyone knows the piano and its derivatives, grand piano, upright piano. But we thought it would be fun to look at other instruments that also have keyboards.
Starting with the mechanical keyboards
The world comes from the french word meaning “heavenly”. It sound is similar to a glockenspiel but softer and more refined. It looks like an upright piano with four octaves. Hammers, operated by the keys, strike metal plates to give that heavenly sound.
These instruments were small in size, compared to pianos and so weren’t very loud. The sound is produced by blades striking iron or brass strings. Very popular in the 16th,17th and 18th centuries
Looking much like a piano, the harpsichord plucks rather that strike the string. Which means that unlike a piano where the pianist can play both loud and softer sounds, the harpsichord, plays with one intensity, no matter how hard one strikes the key.
This a small organ where the player operates foot pedals to pump air through the instrument. Thin metal reeds produce the sound via a keyboard, which controls the airflow.
A hand held instrument, the accordion comprises of a set of bellows which blow air over a set of metal reeds which vibrate. The player flexes the instrument which generates the sound. The keyboard down one side plays the melody, the buttons on the other side plays the base.
These use a keyboard as electronic switches. The switching is processed using microprocessors and converted into sound through a speaker, or recorded by electronic means.
Sounds or samples can be altered so that many variations are possible. Synths can mimic other instruments or natural sounds, such as whale song. Alternatively completely new sounds can be invented and played through the synth.
Just as with violins so too grand pianos have a rich heritage of master craftsman. Here we list some of them and a brief overview of their various histories.
Although many try to list the top ten or top twenty best grand pianos, a sort of pop chart for these mighty expensive instruments. It’s quite a difficult challenge. The truth is that there is no “best” or “worse”and it all comes down to personal preference and even the piece being played. Each of the manufacturers listed here is a grand master we need say no more than than that. So the listing here is in no particular order all make stunningly good pianos and it would be an honor and pleasure to own any one of their instruments.
Steinway and Sons
Founded in 1853 and located in both Hamburg, Germany and New York, United States. These are seen the world over in concert halls everywhere. Each is produced by a master craftsman and each one is unique.
Founded in 1853 and based in Berlin, Germany. Famous for setting up Wigmore hall, a concert in London where their showroom was based. A popular piano for famous rock bands.
Building pianos since 1887, Hamamastsu, Japan. Somewhat of an entry level piano but very well made and a great asset to the world of pianos as they sound good and are most affordable and a great place to start one’s journey.
Started in 1828 in Vienna, Austria. Famous for adding an extra octave to one of their ranges giving a total of 97 keys
Founded in 1978, in Sacile, Italy. Heavy and lavishly decorated, these pianos don’t come cheap. But they are exquisitely well made and are of the highest quality. They’re also a wise investment as not to many get made.
Stuart and Sons
Based in Newcastle, Australia. Somewhat of a newcomer. Their pianos are made to order and they make their pianos using rare and beautiful woods that just add to their appeal. They sound fantastic too.


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Dallas continues to be slighted by culinary bigwigs. This time, Dallas has narrowly missed getting hit by a cream pie thrown by New York fine food arbiters - and that's not a good thing. Said pie landed smack dab in the middle of Paris Coffee Shop in Fort Worth, the venerable diner that was recently named one of Bon Appetit's top 10 places in the U.S. to get a slice of pie.
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We're happy to see a Texas eatery on the best pie list. What we want to know is where to find great pie right here in Dallas. Would Bread Winners make the list? We think so. What about Kathleen's Sky Diner? Absolutely. Would you nominate Mama's Daughter's Diner, Original Market Diner and/or Norma's Café? Tell us where you go to get your key lime, lemon meringue or apple fix. (And, while we're talking apples, consider this smoldering, secondary question: Is cheese the ideal pie pairing?)

domain: food


text:
REPLXENDIANFAIL, SSSS side encountered error while doing endian conversion at journal sequence number JJJJ
Error: The originating or the replicating instance in a cross-endian replication environment report this error whenever they detect that the endian conversion failed.
Action: Restart replication - if the transmission caused the problem, it's probably intermittent. Perform a MUPIP JOURNAL -EXTRACT -DETAIL on the journal files and search for the sequence number JJJJ to look for anything different about that journal record. If the report is on the secondary, take a fresh backup on the originating instance requesting new journal files, refresh the replicating instance and restart replication.

domain: library


text:
Proluton Depot 250 mg (Hydroxyprogestrone Caproate)
Pack contains: Injection 250mg
About Proluton Depot:
Hydroxyprogesterone Caproate is reduce the chances and risk of having a premature baby delivery. The main ingredient of Proluton depot is Hydroxyprogesterone which is manufactured synthetically. Women around the world use this drug to reduce premature delivery.
Uses of Hydroxyprogesterone Caproate:
It is used to reduce the risk of having a premature baby delivery which takes place in 36 weeks or less. In women with one baby or who have earlier delivered a premature baby.
Hydroxyprogesterone caproate is a hormone, used to reduce the risk related to having a premature baby delivery. Premature delivery happens due to lowering of Progesterone levels in women, which is the most important to maintain pregnancy.
Proluton Depot thus helps in maintaining the level of progesterone and prevent premature labor (delivery in under 36 weeks of pregnancy.
Manufacturer of Hydroxyprogesterone Caproate:
The drug is available in the form of two strengths:
- Proluton Depot: 500 mg/200 ml
- Proluton Depot: 250 mg
What to do before taking Proluton Depot:
Before taking this drug or initiating the therapy you should consult your gynaecologist. Patients having diabetes should examined regularly. If you are having any pain around upper abdominal area when you are under this drug then get it checked by your doctor ASAP. You might be having enlarged liver or liver tumour.
Dose of Hydroxyprogesterone Caproate:
You should follow as required or prescribed by your doctor. Regularly visit your doctor for examination and test.
Proluton Depot is injected directly into into the IM. There might be some short lasting reaction like coughing, breathing issues. To reduce these chances you should inject the solution slowly.
For how long I need to take it?
You have to take as long as your doctor asked you to take it. Follow his direction and prescription strictly.
Contact your doctor to know the correct time length of your whole treatment. As it is dependent on the progress you make. Ask your gynaecologist to maintain your progress report.
I missed my dose, what to do now?
If you missed your dose, take your dose immediately when get to remember about it. Never take double dose even if its time to take the next dose.
If you think so may have any issues after taking the medication, contact your doctor immediately. No effect found yet.
- Store your medicines away from direct sunlight.
- Keep under cool and dry place.
- Keep them away from your children.
- If you are taking diabetes medication or insulin, it can change the effect of the medication tell your doctor prior taking Hydroxyprogesterone Caproate
To buy Progesterone Medication , visit: https://www.vaccimedpharmacy.com
|1 ml in 1 Ampoule/s
Proluton Depot 250 mg

domain: medical


text:
Choose apartment Neli as the ideal place for your holiday!
We are located in the city center, only 30m from the palace of the poet Petar Hektorovic .. The apartment is located on the ground floor of the house in front of which you have a parking spa
We'll do our best to make you feel comfortable in your apartment.
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domain: travel


text:
Hawaii Life Real Estate Brokers is certainly feeling the Christmas spirit.
On Monday, the Kauai-based residential real estate firm donated $30,000 to the Hawaiian Islands Land Trust’s “Founding Few” program.
“We’re honored to contribute, and we’re even more honored to take a stand on behalf of the Hawaiian Islands Land Trust and ... encourage you to support them as well,” Hawaii Life Real Estate Brokers co-owner Matt Beall said on his company’s blog. “As with any fundraising, it’s not the amount that you give, it’s that you give.”
Founded in 2011, the Hawaiian Islands Land Trust is a nonprofit committed to working with private landowners, community groups and leaders, as well as government partners, to protect the state’s abundant natural resources.
“If you’ve spent time in Hawaii, you’ve already benefited from the protection of shorelines and open space that the Land Trust has helped to provide,” Beall said. “When we started Hawaii Life, we knew we wanted the company to be committed to philanthropy, [and] we’re strong believers in supporting our communities, the islands where we live, and all of Hawaii.”
Hawaii Life Real Estate Brokers, which is based in Hanalei, currently has about 140 agents and seven offices across the state.

domain: news


text:
PC game players had a smorgasbord of blockbusters on the menu in 2023, with so many bright, shiny worlds ready to explore. While there were some consoles exclusives that skipped PC this year – notabley titles like Marvel’s Spider-Man 2 and Final Fantasy 16 – the vast majority of games that launched were also available on PC.
That what makes this year’s Best PC Games GOTY list so difficult to curate. There were genuinely so many great games on PC this year, and such a vast, diverse array of titles. From the wilds of Baldur’s Gate 3 and Alan Wake 2, to the dark, twisting imagination behind Lies of P, there was so much to experience – and that’s not even scratching the surface of all the unique genres that only exist on PC.
It’s taken a long time to narrow down our favourite PC picks from what we’ve played this year – but after much deliberation, we’ve finally arrived. Here’s the GamesHub list of the best PC games in 2023.
How does GamesHub pick its Game of the Year list?
GamesHub’s Game of the Year picks are selected collectively by tenured staff and regular contributors. Each participant puts together a ranked personal list of their favourite games released in 2023, and titles are given a score according to their rank, with 10 being the highest, and 1 being the lowest. The scores are collated, and the games are ranked by their collective score, with staff members adjusting where necessary before locking in the final list.
Table of Contents
To kick off, we should mention our starry array of honourable mentions – all the PC games we loved in 2023, but ones that didn’t quite make our top five list. Let’s kick off with Venba, an excellent narrative game about family, cooking, cultural connections, and also with Shadows of Doubt, an incredibly innovative detective game. Both were made by smaller studios, and each had a wonderful impact this year.
Honourable mentions should also be given to blockbuster space adventure Starfield, for its unique approach to space travel, Cyberpunk 2077: Phantom Liberty for its snazzy spy missions, Resident Evil 4 for its terrifying approach to horror survival, Knuckle Sandwich for its Aussie-infused RPG ingenuity, and to Street Fighter 6, for its wonderfully silly story mode, and robust battles.
And now, onto our list of the top five best PC games in 2023.
Baldur’s Gate 3
Baldur’s Gate 3 is an incredible role-playing experience that seems to change shape based on individual players. Nearly all of my friends became Baldur’s Gate 3 stans in 2023, and we all played the game in tandem, making for an ultra confusing time when we frequently wound up in different areas, meeting different people, and experiencing different events. I will never forget when someone told me they’d just fought dinosaurs, and I had zero clue how that was even possible.
In true Dungeons & Dragons fashion, it’s a game that gets incredibly wild, and one filled with wacky, wonderful moments at every turn – whether you choose to indulge in mind worms, romance a Mind Flayer, piss off a genie, find the pieces of a dead clown, or otherwise spend time actually playing through the game’s sweeping plot. No matter what you choose, you’re in for some real gold. – Leah J. Williams
Lies of P
- See Lies of P on Amazon.
For all the games inspired by Dark Souls, Bloodborne, and the rest of the FromSoftware repertoire, I’ve never seen one that has completely enamoured people like Lies of P.
Perhaps it’s the stunning art direction, featuring Victorian costumes and creatively macabre mechanical terrors. Perhaps it’s the inventive reframing of the story of Pinocchio, and how the game uses it to do something interesting in examining themes of self-actualisation. Or perhaps it’s the tight and exciting combat, featuring inventive weapons and an entertaining array of prosthetic limbs, whose abilities can completely change the way you approach things.
Whatever it is, it’s clear the developers at NeoWiz have been able to tap into what makes the Soulslike genre resonate so strongly, and have excelled at putting their own unique spin on things. If you’re a Souls fan, do not sleep on Lies of P, and keep a close eye on this studio. – Edmond Tran
Armored Core VI: Fires of Rubicon
Even more so than its previous games, FromSoftware’s Armored Core 6: Fires of Rubicon makes every major victory against its overwhelming odds, and its fearsome boss characters, feel exhilarating, well-fought, and hard-earned. In a desolate far-flung future where industry mars the world, corporations are at each other’s throats, and alliances change on a whim, it feels like there is no hope. But that’s what makes charging through the awfulness feel so liberating.
No RPG levelling, no hand-holding – it’s just you and the mech you built against the world. If you fuck up, maybe you change out your parts, your weapons. But at the end of the day, it comes down to you and your persistence, your focus, and your mettle. It’s a power fantasy you have to work hard for. When you hear the crunch of metal and see the dramatic slow-motion effect when your killing blow lands… it feels like you could punch the moon. – Edmond Tran
- See Dredge on Amazon.
What a vibe. What a loop. Unknown horrors lurk just below the surface in Dredge, just out of view, just out of reach. That tantalising ‘almost’ feeling is intoxicating, and permeates every facet of this horror fishing game. A fishing game! We’re talking about a fishing game here, folks. How can a fishing game be so engaging? So unnerving? So moreish?
It’s that habitual feeling of wanting to try and catch just one more fish before darkness covers the ocean, and you start to see scary things. To sail out just a little bit further to reach the next safe haven across rocky waters. You’re constantly pushing yourself to the brink for that extra bit of a gain, or to get a peek at what’s through the veil – even if in the back of your head, you really don’t want to. Dredge is a masterclass in atmosphere that gets stuck into your brain. It’s an unforgettable journey with some seriously fucked up fish. – Edmond Tran
Alan Wake 2
Alan Wake 2 positively blew me away in 2023, after more than a decade of waiting for it. Frankly, Remedy Entertainment outdid itself with this sequel. It’s creepy, and gorgeous, and extremely obtuse, in a way that’ll have you salivating for answers, generating conspiracy theories, and going deeper into its strange, winding lore. Remedy is a company known for its storytelling, and Alan Wake 2 represents the best of what the studio is capable of.
It’s a multi-threaded monster. A literature-minded horror adventure with sprawling plot threads that dangle and tease, inspiring questions, curiosity, and an ever-forward march as it spins a complex web of terrors. But while it weaves a tale that seems to dance out of reach with every strange chapter, as the interlocking stories of Saga Anderson and Alan Wake play out, deft writing and a strong plot means the answers eventually coalesce in gripping fashion.
That’s not to mention that Remedy hid a full-blown musical in this game, and plenty of other quirks besides. It’s a phenomenal experience and one well worth exploring yourself. – Leah J. Williams
Catch up with more of GameHub’s favourite games of 2023, including platform-specific lists and picks from special guests in our Best of 2023 roundup.
GamesHub has affiliate partnerships. These do not influence editorial content. GamesHub may earn a small percentage of commission for products purchased via affiliate links.

domain: entertainment


text:
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text:
We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.
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Mac-Daniel, L., Buckwalter, M. R., Gueirard, P., Ménard, R. Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites. J. Vis. Exp. (111), e53796, doi:10.3791/53796 (2016).
Translate text to:
Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.
Malaria is one of the deadliest infectious diseases in the world, killing more than half a million people per year. Infection by Plasmodium, the causal agent of the disease, begins by a pre-erythrocytic (PE) phase. During this phase, sporozoites injected into the host skin by a female Anopheline mosquito reach the liver via the bloodstream and differentiate inside hepatocytes into the parasite forms that infect red blood cells and cause the symptoms of the disease.
The PE stages of Plasmodium represent a privileged target for anti-malaria vaccination. Indeed, live attenuated vaccines against these stages, such as radiation attenuated sporozoites (RAS), genetically arrested parasites (GAP) or chemoprophylaxis and sporozoites (CPS) have demonstrated their capacity to protect both rodent and human hosts1-9. In the rodent model, most vaccination studies are conducted utilizing intravenous immunization, which is the gold standard in terms of protective efficacy. However, the description of a skin stage and the importance of the skin-associated draining lymph node (dLN) in eliciting protection has changed our perception of the PE phase and emphasized the importance of the intradermal route of injection. Intravital imaging of P. berghei sporozoites injected into the skin of rodents has shown that only ~25% of the inoculum reaches the liver via the bloodstream. The remaining ~75% distributes between the proximal dLN (~15%) and the skin (~50%)10,11, where a small proportion can transform and remain alive for weeks inside skin cells12,13. Moreover, subsequent studies described that the establishment of effective protective immunity after intradermal immunization mainly takes place in the skin-dLN, where parasite specific CD8+ T cells are activated, and only marginally in the spleen or liver-dLNs14,15.
While most studies have concentrated on the characterization of the effector cells implicated in the establishment of protective immune response, much less is known about the fate of live attenuated parasites injected into the skin, especially their interactions with the innate immune system. In particular, characterization of antigen-presenting cells involved in parasite antigen uptake, processing and presentation to CD8+ T cells is of critical importance, knowing that PE antigen acquisition can occur both in the skin and dLN compartments. Previous intravital imaging studies described an early influx of brightly fluorescent Lys-GFP positive cells in the skin following an infectious mosquito bite16 while early interactions between sporozoites and dendritic cells were observed in the dLN10,17. More recently, it has been reported that sporozoites inoculated in the skin by mosquitoes increases the motility of both dendritic and regulatory T cells in the skin of mice, while a decrease number of antigen presenting cells was observed in the dLN18.
We aimed to identify and quantify more precisely the leukocyte subsets recruited in the skin and corresponding dLN as well as those interacting with the parasite following intradermal injection of immunizing doses of RAS19. In this context, we isolated myeloid cells (CD45+CD11b+) from both tissues and characterized subpopulations of interest by multi=parametric flow cytometry. Consistent with the immune response described in the early stage of Leishmania major skin infection20, the primary host response to sporozoite injection consists of a successive recruitment of polymorphonuclear neutrophils (CD45+CD11b+Ly6G+Ly6Cint) followed by inflammatory monocytes (CD45+CD11b+Ly6G-Ly6C+) which are identified on the basis of differential expression of the Ly6G and Ly6C surface markers.
We describe here a protocol for isolating myeloid cells from the mouse skin and dLN following intradermal injection of immunizing doses of RAS extracted from infected mosquito salivary glands. Reproducible intradermal injections and tissue processing are critical steps to quantify phenotypic changes of infiltrating cell population within infected tissues. The approach detailed below provides a reliable assay to assess the skin and dLN inflammatory response to Plasmodium parasite and can be extended to various experimental systems.
All procedures were approved by the committee of the Pasteur Institute and by the local Ethics Committee on Animal Experimentation (Ethical committee IDF-Paris 1, Paris, France; agreement number: 2012-0015) and performed in accordance with the applicable guidelines and regulations.
1. Materials and Reagents
- Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously21.
- Use parasites Plasmodium berghei ANKA clone expressing the Green Fluorescent Protein (GFP) gene under the control of the constitutive Heat Shock Protein 70 (HSP70) promoter. This yields bright fluorescence throughout the parasite life cycle22.
- Use female C57BL/6JRj mice (7 week-old).
NOTE: All the reagents used in the described protocol are listed in the Table of Materials/Equipment.
2. Radiation-attenuated Sporozoite Isolation from Mosquito Salivary Glands
- Between 18-25 days after the infectious blood meal, collect and cold-anesthetize mosquitoes into a 15 ml tube on ice as described previously21. Carefully transfer them onto a Petri dish by inverting the tube. Maintain the insects on ice and expose the mosquitoes to a 12 krad dose of γ- or x-irradiation.
- Isolate attenuated sporozoites from irradiated mosquito salivary glands as previously described21. Collect infected salivary glands in a small volume (around 15 μl) of 1x Dulbecco's phosphate-buffered saline (DPBS) on ice and gently crush them to release sporozoites.
NOTE: The injection of a highly concentrated parasite suspension in a small volume being critical, it is therefore essential to harvest a sufficient number of salivary glands from a high number of preselected well-infected mosquitoes, as evidenced by their robust expression of green-fluorescence.
- Filter the sporozoite suspension through a 35 µm cell strainer cap adapted to a low adhesion 1.5 ml microcentrifuge tube in order to eliminate clumps and mosquito debris.
- Count the total number of isolated sporozoites as previously described21 and adjust the concentration of parasite suspension with cold 1x DPBS to obtain 83,000 sporozoites/μl. Store the parasites on ice while continuing the next steps.
- Collect the equivalent number of salivary glands from uninfected mosquitoes that will be used as negative control and process the sample as described in steps 2.2 and 2.3. Dilute the sample as indicated above to obtain similar concentration of salivary gland extracts in the suspension.
NOTE: Sporozoites can be stored on ice for a maximum of 2 hr. However, ideally they are injected within an hour after crushing the salivary glands.
3. Injection of Sporozoites into the Dermal Layer of the Ear
- Anesthetize animals by intraperitoneal injection of ketamine (50 mg/kg)/xylazine (5 mg/kg) mixture. Wait 5-8 min for the mouse to be in a state of unconsciousness and apply ophthalmic ointment to the eyes to prevent corneal drying. Evaluate the depth of anaesthesia by performing a gentle toe pinch on both rear feet.
NOTE: The dose of anaesthesia lasts for less than 20 min, so the next steps must be done quickly.
- Stabilize the ear pinna of the mouse by fixing the ventral side with clear tape previously placed under a stereomicroscope (Figure 1A). Gently press the ear to avoid damage of the vasculature.
- Load a 10 μl syringe/35 gauge bevelled needle with 0.6 µl of resuspended parasites10.
- Deliver the sporozoite suspension in 4 injection sites (0.15 µl per site) by carefully inserting the needle on the dorsal side of the ear (Figure 1A), beneath the epidermis, with the bevel up, taking care to avoid any blood vessels. Allow the needle to remain in the dermis for few sec to prevent backflow of the injectant before removing it. Observe a characteristic papule at each injection site at the end of the procedure (Figure 1B and 1C).
- After injection, carefully remove the ear from the tape.
- Inject the contralateral ear with the same dose of parasites by following the steps 3.2-3.4.
- Inject 2 additional mice with either 0.6 µl of 1x DPBS or 0.6 µl of 1x DPBS + salivary gland extract from uninfected mosquitoes in order to evaluate the inflammatory response mediated by the injection alone and the deposition of mosquito material in the dermis.
- Monitor mouse recovery from anaesthesia before placing it back into the cage.
NOTE: The proper sporozoite deposition in the skin can be monitored by fluorescence microscopy (Figure 2A and 2B), the mouse being prepared as described previsously23.
4. Skin and Draining Lymph Node Dissection
- Prepare standard medium (SM) as follows: Dulbecco's Modified Eagle Medium (DMEM) + 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplemented with 400 U/ml collagenase and 50 μg/ml deoxyribonuclease (DNase).
- Prepare two 6 well flat-bottom tissue culture plates on ice with 4.5 ml SM + 70 μm cell strainer per well for dLN samples (Plate A) and 4.5 ml SM per well for skin samples (Plate B).
- At the desired time point following sporozoite inoculation, deeply anesthetize the mouse by intraperitoneal injection of ketamine (125 mg/kg)/xylazine (12.5 mg/kg) mixture prior cervical dislocation. Make sure the mouse demonstrates signs of clinical death and start the dissection immediately.
NOTE: The kinetics of myeloid cell recruitment can be examined at different times after injection (i.e. 2 hr, 4 hr and 24 hr as previously described14).
- Disinfect the inoculated ear and the corresponding neck region with 70% ethanol to reduce the possibility of contamination.
- Using sterile scissors and forceps, aseptically dissect the proximal auricular dLN without collecting the surrounding fat. Perform a first incision in the jugulo-carotidian sulcus and remove the hairs from the operating field. Stretch the incision with 2 forceps to expose the dLN that appears grayish compared to the surrounding tissue.
- Pinch the connective tissues carefully on the top of the dLN with forceps to facilitate its removal. Extract the dLN by placing a forceps underneath the lymphoid tissue (Figure 3A and 3B). Place the dLN in a well containing 4.5 ml SM + 70 μm cell strainer (Plate A).
- Harvest the entire inoculated ear using sterile sharp scissors and forceps (Figure 4A). Separate the dorsal and ventral aspects of the ear by pinching and spacing apart the cut ends with two forceps (Figure 4B-4E). Place them into a well containing 4.5 ml SM (Plate B) making sure both tissues are completely immersed in the medium.
NOTE: In order to improve the reliability of the experiment and increase the number of cells of interest, pool 2 injected ears or 2 dLNs from the same mouse per sample.
- Harvest and process in parallel the ears and dLNs of mice inoculated with either 1x DPBS or salivary gland extracts. Include 2 additional ear and lymph node samples harvested from a naive mouse for negative and single staining compensation controls.
5. Cell Isolation from Lymph Nodes
- Incubate the lymph nodes (Plate A) at 37 °C + 5% CO2 for 15 min under gentle agitation. Add 10 mM final Ethylenediaminetetraacetic acid (EDTA) per well to neutralize collagenase and DNase activities. Perform the next steps on ice.
- Dissociate lymph nodes using the top end of a 5 ml syringe plunger. Press in circular motions against the strainer until all that remains is white connective tissues.
- Rinse the cell strainer twice with 500 μl 1x DPBS + 2% Fetal bovine serum (FBS) + 2mM EDTA. Transfer entire volume of the well in a 15 ml tube.
- Rinse the well twice with 500 μl 1x DPBS + 2% FBS + 2mM EDTA and transfer the volume into the 15 ml tube. Keep tubes on ice.
6. Cell Isolation from Skin of the Ear
- Incubate the ear leaflets (Plate B) at 37 °C + 5% CO2 for 1 hr under gentle agitation.
NOTE: After 20 min of incubation, cut the ear leaflets with scissors into small pieces to facilitate tissue digestion and put the sample back in the incubator for the remaining 40 min. Single cells can be observed in the medium at the end of the incubation time.
- Add 10 mM final EDTA per well to neutralize collagenase and DNase activities. Perform the next steps on ice.
- Transfer the ear tissue fragments and the whole volume of medium in a new well containing a 70 μm cell strainer using a 1ml pipette. Cut 2 to 3 mm from the end of the tip to collect the ear tissue fragments more easily.
- Dissociate ear tissue fragments using the top end of a 5 ml syringe plunger. Press in circular motions against the strainer until all that remains is white connective tissues.
- Rinse the cell strainers twice with 500 μl 1x DPBS + 2% FBS + 2 mM EDTA.
- Transfer the entire volume of the well into a 50 ml-tube through a 70 μm cell strainer.
- Rinse the well with 500 μl 1x DPBS + 2% FBS + 2 mM EDTA and transfer the volume into the 50 ml tube. Keep the tube on ice.
NOTE: The different steps to isolate total cells from the skin and dLN tissues are summarized in Figure 5.
7. Count and Viability of Collected Cells
- Centrifuge the skin and dLN samples for 5 min at 450 x g at 4 °C and discard the supernatant. Gently resuspend the pellet with 300 μl 1x DPBS + 2% FBS + 2 mM EDTA.
- Collect a small volume of each sample to count the total number of cells isolated from the skin and dLN using a hemocytometer. Add 0.04% trypan blue to determine the cell viability.
8. Blocking of Non Antigen-specific Binding
- Add 10 µg/ml of unlabelled CD16/CD32 Fc-block antibody. Incubate 20 min at 4 °C (can go longer).
- Add 2 ml of cold 1x DPBS + 2% FBS + 2 mM EDTA. Centrifuge the sample for 5 min at 450 x g at 4 °C and discard the supernatant. Gently resuspend the pellet with 300 μl 1x DPBS + 2% FBS + 2 mM EDTA and maintain the samples on ice.
9. Staining Skin and Lymph Node Myeloid Cells
- Incubate previously blocked skin and dLN isolated cells with Live/Dead tracker (i.e. 4',6-diamidino-2-phenylindole or DAPI), anti-CD45 and anti-CD11b specific antibodies.
NOTES: In order to enrich the myeloid cell subpopulations of interest, especially rare ones, CD45+ cells and lymphocytes can be respectively purified from the skin or depleted from the dLN by magnetic bead facilitating cell sorting. Since most of the cells isolated from the dLN are CD45+, the use of anti-CD45 is not mandatory. Other markers can be included to identify myeloid cell subpopulations of interest by positive or negative-gating strategy. In this protocol, the myeloid cells recruited in the dLN were enriched by excluding CD8α+ cells (the whole T cell compartment can be targeted by using anti-CD3 specific antibody).
- Incubate 30 min at 4 °C in the dark. Add 500 μl 1x DPBS + 2% FBS + 2 mM EDTA, centrifuge the sample 5 min at 450 x g at 4 °C and discard the supernatant. Repeat the wash step twice.
- Resuspend the samples with 300 μl 1x DPBS + 2% FBS + 2 mM EDTA. Transfer the entire volume in a FACS tube through a 35 µm cell strainer. Rinse the filter with 100 μl 1x DPBS + 2% FBS + 2 mM EDTA.
- Run the stained cells in a flow cytometer. Gate live single cells (DAPI-,FSC-W) to exclude dead cells and doublets. Plot CD45+CD11b+ events for the skin (Figure 6A) and (CD45+)CD8α-CD11b+ events for the dLN (Figure 6B).
We recently demonstrated that needle-syringe injection of immunizing doses of P. berghei sporozoites in the mouse skin induces a successive recruitment of polymorphonuclear neutrophils followed by inflammatory monocytes in the skin and dLN19. The Protocol Section described above details the procedure used to successfully isolate live myeloid cells from both tissues following multiple injections of large number of sporozoites in the ear dermis (Figures 1 and 2). Within the first 24 hr, the dLN (Figure 3) and the skin injection site (Figure 4) were isolated and processed as summarized in Figure 5 to analyse the cellular infiltration by multi-parametric flow cytometry. Overall, this technique allowed us to isolate an average 104 CD45+CD11b+ and 2.104 CD11b+ live single cells for the skin and dLN respectively. The acceptable viability of non-debris single cells ranged from 40-70% for the skin and 70-90% for the dLN. As shown in Figure 6, the frequency of myeloid cells in the skin (CD45+CD11b+) and the dLN (CD8α-CD11b+) strongly increases following intradermal injection of RAS compared with non-infected mice.
Figure 1: Intradermal Injection of Sporozoites into the Ear of Mouse. (A) Image showing intradermal injection of sporozoites in the ear pinna of an anesthetized mouse. The ventral side of the ear is stabilized with clear tape previously placed under a dissecting microscope. The needle is carefully inserted on the dorsal side of the ear, beneath the epidermis, with the bevel up. (B-C) Pictures showing the dorsal side of the ear pinna prior (B) and after (C) intradermal injection of 0.1-0.2 μl of parasite suspension. A characteristic papule (black arrow) is observable at the injection site at the end of the operation. Please click here to view a larger version of this figure.
Figure 2: Imaging of Sporozoites Deposited in the Ear Dermis of Mouse. (A) Fluorescence microscopy showing RAS migrating from the injection site (indicated by the dashed lines) in the mouse skin 15 min post-injection (Scale bar = 60 μm; 10X magnification). The path of sporozoites is represented by the maximum intensity projection of the fluorescent signal. Green and red fluorescence respectively show parasite position in the skin at the beginning and after 10 sec of acquisition. (B) Higher magnification of RAS (green) injected in the skin (Scale bar = 20 μm; 25X magnification). Please click here to view a larger version of this figure.
Figure 3: Draining Lymph Node Processing. (A) Picture showing the dissection of the proximal auricular dLN following intradermal injection of Evans blue for demonstration purposes. After cervical dislocation of the mouse, the neck is disinfected with 70% ethanol. A first incision is made in the jugular-carotid area with sharp scissors and the skin is removed from the operating field. The incision is stretched with 2 forceps to expose the dLN that appears grayish compared to the surrounding tissue. The connective tissues are then carefully pinched with forceps on the top of the dLN and progressively separated from it. Finally, the dLN is extracted by placing a forceps underneath the lymphoid tissue. (B) Picture showing the extracted dLN in a drop of PBS. Please click here to view a larger version of this figure.
Figure 4: Ear Skin Processing. Pictures showing the successive steps to process the inoculated ear. (A) After cervical dislocation of the mouse, the ear is disinfected with 70% ethanol and removed using sterile sharp scissors and forceps. (B-D) The dorsal and ventral aspects of the ear are lightly separated by pinching and spacing apart the cut ends with two forceps. (E) Picture showing the dorsal and ventral aspects of the ear prior enzymatic treatment. Please click here to view a larger version of this figure.
Figure 5: Protocol Summary. Schematic representation of the key steps to isolate total cells from the skin and dLN tissues. Please click here to view a larger version of this figure.
Figure 6: Myeloid Cell Population Isolated From the Ear Skin and the Proximal dLN. Representative FACS plots showing the gating strategy to analyse the myeloid compartment in the skin (A) and the dLN (B). CD45+CD11b+ and CD8α-CD11b+ cells were gated on total live singlet events for the skin and dLN respectively. For each condition, 5 x 105 total events were acquired (2 ears and 2 dLN pooled per sample). Please click here to view a larger version of this figure.
In the perspective of large-scale vaccination of humans using a whole sporozoite malaria vaccine, one of the main challenges to overcome is to develop optimized routes and methods of parasite administration to ensure successful immunization and protection24,25. In humans, the evaluation of protective efficacy mediated by live attenuated parasites (LAP) has been performed following natural mosquito bites2, as well as intradermal, subcutaneous25,26 and IV immunizations27. As reported in rodents28 and non-human primates25, IV administration of LAP induces stronger protective immune responses compared to the above-mentioned routes. Nevertheless, non-IV routes of administration using needle and syringe still remains more suitable for human mass vaccination and efforts are currently being made to optimize their protective efficacy.
Recent studies conducted in mice demonstrated, especially in the case of the ID route, that the deposition of a small volume of sporozoite suspension (range of 1 µl) in multiple injection sites (4 points) significantly increased parasite infectivity compared to a single inoculation of parasites diluted in larger volumes (50 µl)24. In our experimental system (C57BL/6 mouse - P. berghei), complete protection was achieved after multiple injections in the ear dermis of highly concentrated suspension of parasites (50,000 RAS in 0.6 µl, 4 injection sites)19. In this context, we established the protocol described above to analyze more precisely the local host immune response to immunizing doses of RAS that allowed us to identify 2 major myeloid cell subsets recruited in the skin and dLN in response to the parasite19.
Multi-parametric flow cytometry allows accurate identification and quantification of immune cell changes in the context of host response to infection19,20. Isolation of large number of viable cells is therefore critical to perform reproducible phenotypic analysis. For this purpose, the enzyme concentration and the duration of tissue digestion must be optimized. Indeed, low concentration of enzymes or insufficient incubation time may lead to incomplete digestion with low yield of cells while prolonged tissue digestion may result in decrease cell viability and alteration of cell surface antigen expression29. Nevertheless, well-established protocols using collagenase treatment have been shown to have marginal impact on the detectability of relevant surface molecules frequently used for phenotypic analyses30.
Among the several parameters that can optimize the quality of cell isolation, the separation of the dorsal and ventral sections of the ear skin is essential, since it allows the collagenase/DNAse to access the dermis. In addition, mincing the skin into small pieces increases the surface area accessible to the enzymes, therefore allowing shorter duration of digestion. Finally, the use of mechanical dissociation helps to increase the yield in cell number for both the skin and dLN. However, excessive dissociation can cause additional stress that could negatively impact cell viability.
We favoured acquisition of live cells since fixation leads to suboptimal staining, complicating distinction of rare positive events of interest (data not shown). Moreover, the discrimination of dead cells was more problematic. In cases where cell fixation is required, we would suggest the use of LIVE/DEAD fixable dead cell stains.
Using the defined protocol, we successfully isolated myeloid cells infiltrating into the skin and dLN in response to parasite injection (Figure 6A and B). We usually obtain higher levels of viable single cells from the dLN (70-90%) compared to the skin (40-70%). This difference can be explained by the longer incubation time needed for efficient enzymatic digestion together with additional steps of mechanical dissociation to isolate skin cells (skin layer separation, mincing and stronger mashing).
Finally, an important parameter to take into account when analysing the host response to ID injection of sporozoites is the level of inflammation induced by both needle insertion and mosquito salivary gland extract deposition in the skin19,20. We therefore suggest to inject additional mice with either 1x PBS alone or salivary gland extracts from the same number of non-infected mosquitoes to evaluate the immune response specifically induced by the parasite. Altogether, we advise to dissect infected mosquitoes with high parasite load in the salivary glands and to filter the parasite suspension to limit the amount of mosquito material that could be deposited in the skin.
In summary, the isolation of myeloid cells from the skin and dLN through the described protocol provides a reliable assay to assess dynamic cellular changes during inflammatory response to Plasmodium infection and can be extended to other pathogens transmitted into the skin of the host.
The authors declare that they have no competing financial interests.
The authors thank Patricia Baldacci, Vanessa Lagal and Sabine Thiberge for critical reading, Irina Dobrescu and Sabine Thiberge for help in taking pictures and Pauline Formaglio for teaching in vivo imaging of sporozoite motility in the mouse skin. We also would like to thank Marek Szatanik and the Center for Production and Infection of Anopheles (CEPIA-Institut Pasteur) for mosquito rearing. This study was supported by the AXA Research Fund and funds from the Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (grant no. ANR-10-LABX-62-IBEID).
|Ketamine: Imalgene® 1,000||Merial||-||50 mg/kg: prior sporozoite injection 125 mg/kg: prior sacrifice|
|Xylazine: Rompun® 2%||Bayer||-||5 mg/kg: prior sporozoite injection 12.5 mg/kg: prior sacrifice|
|NanoFil syringe + 35 gauge needle||World Precision Instruments||-||-|
|Omnican® 50 Insulin syringe 0.5 ml/50 I.U.||B. Braun Medical||9151125||-|
|MultiwellTM 6 well tissue culture plate – Flat Bottom||BD Falcon||353046||-|
|70 µm cell strainer||BD Falcon||352350||-|
|2 ml syringe||Terumo||SS-02S||-|
|BLUE MAXTM 15 ml Polypropylene conical tube||BD Falcon||352097||-|
|BLUE MAXTM 50 ml Polypropylene conical tube||BD Falcon||352098||-|
|5 ml Polystyrene Round-Bottom Tube with 35 µm Cell-Strainer Cap||BD Falcon||352235||-|
|DPBS 1x CaCl2- and MgCl2--free||Life Technologies||14190-094||-|
|DMEM 1x + GlutaMAXTM||Life Technologies||31966-021||-|
|Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid||Sigma-Aldrich||C5138||400 U/ml|
|Deoxyribonuclease I from bovine pancreas, type IV||Sigma-Aldrich||D5025||50 µg/ml|
|EDTA disodium salt||Sigma-Aldrich||E-5134||10 mM or 2.5 mM|
|HEPES buffer solution (1M)||Gibco||15630-056||25 mM|
|Trypan blue Stain (0,4%)||Life Technologies||15250-061||Dilution 1:10|
|Anti-mouse CD16/CD32 (2.4G2 clone)||BD Biosciences||553142||10 µg/ml final (1:50)|
|DAPI FluoroPureTM grade||Life Technologies||D21490||1 µg/ml final|
|Anti-mouse CD45 (30-F11 clone)||BD Biosciences||559864||Dilution 1:200|
|Anti-mouse CD11b (M1/70 clone)||BD Biosciences||557657||Dilution 1:400|
|Anti-mouse CD8α (5H10 clone)||Life Technologies||MCD0830||Dilution 1:100|
|Female C57BL/6JRj mice (7-week-old)||Janvier Laboratories||-||-|
- Nussenzweig, R. S., Vanderberg, J., Most, H., Orton, C. Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei. Nature. 216, (5111), 160-162 (1967).
- Hoffman, S. L., et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis. 185, (8), 1155-1164 (2002).
- Mueller, A. K., et al. Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface. Proc Natl Acad Sci U S A. 102, (8), 3022-3027 (2005).
- Mueller, A. K., Deckert, M., Heiss, K., Goetz, K., Matuschewski, K., Schlüter, D. Genetically attenuated Plasmodium berghei liver stages persist and elicit sterile protection primarily via CD8 T cells. Am J Pathol. 171, (1), 107-115 (2007).
- Tarun, A. S., et al. Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells. J Infect Dis. 196, (4), 608-616 (2007).
- van Dijk, M. R., et al. Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells. Proc Natl Acad Sci U S A. 102, (34), 12194-12199 (2005).
- Butler, N. S., Schmidt, N. W., Vaughan, A. M., Aly, A. S., Kappe, S. H., Harty, J. T. Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites. Cell Host Microbe. 9, (6), 451-462 (2011).
- Belnoue, E., et al. Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J Immunol. 172, (4), 2487-2495 (2004).
- Behet, M. C., et al. Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum. Malar J. 13, 136 (2014).
- Amino, R., et al. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat Med. 12, (2), 220-224 (2006).
- Yamauchi, L. M., Coppi, A., Snounou, G., Sinnis, P. Plasmodium sporozoites trickle out of the injection site. Cell Microbiol. 9, (5), 1215-1222 (2007).
- Gueirard, P., et al. Development of the malaria parasite in the skin of the mammalian host. Proc Natl Acad Sci U S A. 107, (43), 18640-18645 (2010).
- Voza, T., Miller, J. L., Kappe, S. H., Sinnis, P. Extrahepatic exo- erythrocytic forms of rodent malaria parasites at the site of inoculation: clearance after immunization, susceptibility to primaquine, and contribution to blood-stage infection. Infect Immun. 80, (6), 2158-2164 (2012).
- CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes. Nat Med. Chakravarty, S., Cockburn, I. A., Kuk, S., Overstreet, M. G., Sacci, J. B., Zavala, F. 13, (9), (2007).
- Obeid, M., et al. Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria. EMBO Mol Med. 5, (2), 250-263 (2013).
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- Radtke, A. J., et al. Lymph-node resident CD8α+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses. PLoS Pathog. 11, (2), e1004637 (2015).
- da Silva, H. B., et al. Early skin immunological disturbance after Plasmodium-infected mosquito bites. Cell Immunol. 277, (1-2), 22-32 (2012).
- Mac-Daniel, L., et al. Local immune response to injection of Plasmodium sporozoites into the skin. J Immunol. 193, (3), 1246-1257 (2014).
- Ribeiro-Gomes, F. L., Peters, N. C., Debrabant, A., Sacks, D. L. Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response. PLoS Pathog. 8, (2), e1002536 (2012).
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domain: biology


text:
Moraline by Cintia Alfonso Fior is a literary effort on diversity and justice that explores an unlikely young hero’s path to understanding a world that seems bafflingly unfair and richer than we notice.
Table of Contents
Moraline feels like an outcast. She can’t, for the life of her, stay quiet during class, and her classmates don’t seem to like her much. But a sudden encounter with a Lakota Chief sets her on a mission to return the Fourth Wind and, with it, the balance to the Earth. What does this even mean? Will she be able to achieve it? Along with newfound partners, Moraline will find her voice and discover that her weaknesses can become her greatest strengths. Will she ever find a place where she belongs?
The stellar feature of this book is its organic approach to diversity. It is, as in life, a spectrum that depends on who’s watching. On the subtle side (from the narrator’s point of view), we have Serenity, who seems like the other girls, but something’s missing in her family. On the obvious ones, we have Edmundo, who is Mexican, and Amani who is Congolese. The best example of Alfonso’s organic approach to diversity is Brandon. Brandon is portrayed as a handsome, popular basketball star, and we only find out about his race much later. This is also an invitation to question our unconscious biases. How do you picture a handsome basketball star? I’m ashamed to admit I never pictured him looking any different from Zac Efron. But Moraline is here to teach us that we should strive to see skin color and other features as another characteristic but not a defining one. For instance, Amani’s main assets are his brain and his dancing skills, not the color of his skin.
Moraline is not the typical hero, either. Even in her climatic moment, her unique quirks show, which will make her more relatable to young readers. We are all in constant construction and discovery, and I find this approach more realistic and healthier than perfect aspirational characters. We get enough of this apparent perfection in social media, and it’s messing with our heads. (https://jedfoundation.org/resource/understanding-social-comparison-on-social-media/)
The book is full of nuggets of wisdom on handling the challenges the modern world presents without ever being preachy. Kids can extrapolate the colonel’s nonviolent strategy to the slugs invading their tomatoes. But the author never dumbs down the narrative by saying this explicitly. This perfect balance between correctness and realness makes Moraline a great read for middle-school-aged kids that’s not too childish for adults to enjoy.
Cintia Alfonso Fior’s well-rounded approach to bullying is crucial for our youth. The backstories of some antagonists are presented as an explanation for their behavior, but not always. Some people are mean as a defense mechanism, some will find redemption, and some will remain mean. This is a valuable lesson on resilience and adaptability.
Alfonso doesn’t shy away from hard subjects that aren’t usually presented in children’s books. Kids need to know the kind of world they are facing and how to change it for the better. The narrative’s not explicitly violent or profane, but it’s never timid. I think kids appreciate being treated as the smart, capable beings they are.
Valuing our ancestor’s wisdom, learning to listen to nature, shining with our uniqueness, and never giving up on fighting for a kinder world are all things Moraline can teach us. And the fact that there’s a fox as part of the team just makes it even more fun.
- Title: Moraline
- Author: Cintia Alfonso Fior
- Genre: Children’s Book
- Theme: Coming of Age, Native American, Adventure, Diversity
- Publication Date: March 1, 2023
- ISBN or ASIN: B0BNQVG6FQ
- Number of Pages: 190
- Minimum Audience Age: 10
(Note: 0=none, 1=a few, 2=considerable, 3=pronounced, 4=excessive)
- Sexual themes: 0
- Religious themes: 0
- Violence, self-harm, etc.: 0
- Crude language, expletives, swearing, etc.: 0
- Other adult themes: 1
Children’s Book Features
- Good role models for younger ones: Not specified by the author
- Moral values: Not specified by the author
- Good social manners: Not specified by the author
- Age-appropriate language and topics: Yes
- Age-appropriate illustrations: Yes
- Gained the approval of a licensed educator or professionals in a relevant field: Not specified by the author
- Based on scientifically-proven data or similar information: Not specified by the author
- Produced through industry-standard research processes: Not specified by the author
- Other relevant information:
- Content: 5 stars
- Writing Style: 5 stars
- Appeal to Target Audience: 5 stars
- Uniqueness: 4 stars
- Editing: 5 stars
- Other factors: 5 stars
- Overall: 4.8 out of 5
For more posts like this, here’s the link: https://thechrysalisbrewproject.com/category/bookish-posts/.
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domain: literature


Often I am asked as a photographer in Paris, which location I prefer for photos. It is so complicated to say as the city offers so many options especially for an engagement shoot. You can of course decide to capture the iconic monuments like the Eiffel Tower or Musee du Louvre. But to me as a local, nothing says Paris like a cafe. If you never been to Paris you could think that what you see in movies or commercials are made up, cliches, made for tourists. You would be wrong: cafes are truly part of our daily life. This is where we eat of course, but also where we meet our friends, we grab an “Aperitif” drink after work, where we do people watching with our best friend to check the latest trends, meet for a first date, break up, and of course make out.
Taking photos during your engagement shoot at a Cafe to me is the most Parisian thing you could do. It makes for the most romantic photos. Plus it doesn’t hurt to take a little break, warm up with a hot chocolate like during this winter shoot with Nesa and Joe.
For this couple who looks straight out of a movie, it was actually their anniversary. Paris has always been special to them and stopping at a cafe for photos was the best way to capture the essence of the city.
Let me show you some of my favorite products for having a perfect engagement shoot


text:
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domain: hobby


Boerne Village Band
The Boerne Village Band, founded in 1860 by German immigrant Karl Dienger, is the oldest continuously active German band outside of the Republic of Germany. In 1991 Mayor Patrick Heath revived the old Abendkonzert tradition in Boerne with a series of summer evening band concerts on Main Plaza. The free concerts were an overnight success and continue to gain popularity with each passing year. Hundreds of enthusiastic listeners attend each outdoor summer concert and 1000+ attend the yearly July patriotic concert.
Over the years the band has received community awards from the Boerne Area Historical Preservation Society, the City of Boerne, Kendall County, and the Greater Boerne Chamber of Commerce. In 1991 the Texas legislature honored the band for “keeping alive German music as part of our heritage.” In 1996 Germany’s most distinguished award for folk music, the Pro Musica Plakette, was awarded to the band by German president Roman Herzog.
In recognition of the 150th anniversary of the band (1860-2010), Mayor Dan Heckler and the Boerne City Council issued an official proclomation honoring the Boerne Village Band for its many years of dedication to the community.


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East Hartford, CT Police
East Hartford Police Department of East Hartford, CT, U.S.A., offers its residents many emergency services, including a rapid response to incidents and control of a
Address 31 School Street
Phone (860) 528-4401
East Hartford, CT Police reviews :
No reviews added yet. Be the first to add a review for the station.

domain: law


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Apple Inc. Chief Executive Tim Cook is in Beijing this week meeting with China’s largest mobile carriers as the company faces a tougher environment in its second-largest market.
Mr. Cook met with China Mobile Ltd. Chairman Xi Guohua on Tuesday, a spokeswoman for the carrier said. Apple’s CEO also met with officials at China Unicom (Hong Kong) Ltd. on Wednesday, a person familiar with the matter said. In a statement, China’s third mobile carrier, China Telecom Corp., said Mr. Cook also met with senior managers, though it didn’t offer details.
Apple representatives in China didn’t immediately comment on the visit.


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You may remember the story of Diane and Sadie. Diane has been down after her loss of Sadie. Yesterday, we got some good news. Diane loves Dobermans and tried to get a dog from Doberman rescue, but wasn’t successful. She found a litter in San Jose, California and picked up her new pup yesterday.
They named her Jette (pronounced like “jet” as in jet plane). Considering that the dog was in the cargo hold for a spell on weather delay for take off, they should have named her “Tarmac”.

domain: pet


Summit Truck Bodies will feature VMAC product lines at its booth at the upcoming World of Concrete Show in Las Vegas.
The companies announced Wednesday Summit’s booth C1727 will feature Summit’s Ford F550 7 Series Service Trucks, one with a VMAC G30 Gas Engine Driven Air Compressor, and one with a VMAC Hydraulic Driven Air Compressor.
“We’re looking forward to being at the World of Concrete Show this January in the Summit Truck Bodies booth,” says Mike Pettigrew, VMAC’s marketing manager. “Almost all fleet managers and truck upfitters struggle with the size and weight of truck-mounted equipment. VMAC designs the most compact, lightweight, and powerful mobile air compressors and multi-function power systems available. VMAC air compressors reduce the size and weight of equipment and power common pneumatic tools such as impact wrenches, jackhammers, pavement breakers, die grinders, and chipping hammers, making them the best solution for World of Concrete attendees.”
VMAC says its G30 Gas Engine Driven Air Compressor is the most affordable gas driven rotary screw air compressor available. It weighs just 205 lb. and produces up to 30 CFM at 100 percent duty cycle. VMAC’s Hydraulic Driven Air Compressor will also be on display in the booth. The VMAC Hydraulic Driven Air Compressor features the most advanced digital control system available, weighs just 150 lb., and produces up to 40 CFM at 100 percent duty cycle, the company says.
“The World of Concrete Show marks the start of VMAC’s 2018 tradeshow season, which will also include ARA’s The Rental Show in New Orleans in February, and NTEA’s The Work Truck Show in Indianapolis in March,” says Pettigrew. “World of Concrete attendees are invited to the Summit Truck Bodies booth where they’ll be able to chat with representatives from both VMAC and Summit about work trucks and work truck equipment.”


Adobe Marketing Cloud Newsletter - A collection of tech blogs, news and jobs from across the web; all in one convenient place.
AEM Podcast is a resource for developers who manage and maintain websites that use Adobe Experience Manager (formerly CQ5). Get CQ5 help with tips, workarounds, and more.
Learn four best practices that use powerful capabilities within Adobe Analytics to help you develop better insights about your customers.
The Adobe Analytics Fall 2017 release will be available October 26, 2017. It includes updates, enhancements and new features
In this post, we'll discuss how to set up configuration values in the AEM Dispatcher to promote clarity and make it easy to compare and sync configuration files
Ask The AEM Expert - Q&A Session with 3|SHARE
ZURICH, Oct. 24, 2017 /PRNewswire/ -- Cognizant (NASDAQ: CTSH) today announced it has entered into an agreement to...
Evolve18 topics span Adobe Experience Manager, Adobe Campaign, Adobe Analytics, and Adobe Media Optimizer.


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Stock futures are slightly lower ahead of the government’s October jobs report.
Economists expect that employers added a net 100,000 jobs in October. More than twice that number is needed to lower the unemployment rate, which has been stuck at 9.1 percent for the past three months. Employers are reluctant to step up hiring because of uncertainty about the pace of growth and weak consumer demand.
Ninety minutes ahead of the market opening, Dow Jones industrial futures are down 52 points, or 0.4 percent, at 11,924. S&P 500 futures are down 8, or 0.6 percent, at 1,249. Nasdaq 100 futures are down 15, or 0.6 percent, at 2,346.

domain: finance


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Brexit is all over the news and opinions are flying in every direction… so just what is the reaction for value investors? We recommend buying and diversifying.
Brexit, which was the publicly voted for United Kingdom referendum to leave the European Union, has thrown the world’s markets into chaos. The UK’s markets in particular are a whirlwind at the moment, and most sectors have seen sharp drops in their stock prices. As The Telegraph explores in this article, the “pound went crashing below $1.30 in early trade, falling by as much as 1.95% to $1.2798 - a level it last hit in June 1985”. This could be a worrying time for investors.
But, as Scott Nursten (CEO, STRIDE) explained in my last blog, value investors should seize this opportunity. In his parting words of our interview, he recommended that investors find "great companies and buy them cheap. If anyone thinks that - in the long term - there won't be a housing market on a small island that is one of the financial power houses of the world... then they're delusional". Reviewing the intricacies of what value investing is, it seems he’s right… but there’s also something to be said for diversification.
Answering Brexit: Buy and Diversify
Why Should I Buy?
In short, because you have a window of opportunity available for buying UK stocks on the cheap… and if you’re ready for a long-term investing game, then you’ll very likely reap the rewards in 5-10 years.
Scott Nursten did the situation such justice in our interview when he said that “everything from pizza, housing and retail is trading cheap. This response assumes that Brits will stop eating food, buying homes and wearing clothes. That just isn't feasible. There will be changes to certain markets as a result of this, but […] I think it's an over-reaction. When coupled with the pound devaluation, it presents a great opportunity”.
Remember that in 1987, 2001 and 2008 global markets plunged by over 30%. People were lost in the chaos. Yet, less than 18 months later in each case, they started to recover. After each plunge, the markets had recovered fully within 5 years. But, those stats only reflect indices. Just imagine the valuable returns with effective stock selection and diversification. Brexit is offering a similar situation.
Why Should I Diversify?
If you follow STRIDE’s 3D value investing techniques, you’ll know that diversification is always recommended. But, it’s particularly recommended in volatile times (which Brexit certainly is).
Diversification should be used to mediate risk. It helps to spread your investments over multiple sectors, industries and currencies, essentially limiting your risk. How? By spreading your eggs between many baskets. You only ever diversify from an understanding that we can’t control markets, and that they will always fluctuate. We can’t stop a market’s fall, but we can protect ourselves against it.
In order to diversify well, you’ll need to:
- Understand what your investment goals are (short-term, long-term, or a combination) by completing your investor’s profile.
- Decide at what level of risk you’re willing to invest into (create your investor's profile).
- Analyse which stocks, properties or markets match your investor’s profile.
- Once you’ve selected your investments, divide them between their risk factors (market cap, industry, geography, etc.).
The remarkable thing about a well-diversified portfolio is that where one stock might fall short, another will likely rise to make up the loss. There is always the possibility of risks that are harder to diversify (such as Brexit, the US elections or geological disasters), but if you’re protected against more general risks, these won’t have quite the same negative effect.


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Events and Concepts
Used to understand the novel Of Mice and Men
Created By: Alex Gonsalves
The Great Depression
The Great Depression, which lasted from 1929-1939, was the longest lasting economical downfall in the history of the Western world. The event began shortly after the crash of the stock markets in October of 1929. The stock market crash made Wall Street panic and wiped out millions of investors. Consumer spending and investment declined quickly over the next couple of years, causing less industrial output and unemployment levels rising as many of the companies laid off employees. When the Great Depression had hit its lowest point in 1933, 13 to 15 million Americans were unemployed and almost half of the country’s banks had failed. Even though at the time President, Franklin D. Roosevelt, helped lessen the effects of the Great Depression, the economy wouldn’t turn completely around until 1939.
A program that aided in the recovery from the Great Depression was the Tennessee Valley Authority, or TVA. To help, they built dams and hydroelectric projects to control flooding and provide electric power to the poor town. The Works Project Administration was a permanent jobs program that employed over 8.5 million people between the years of 1935-1943. The issue was finally resolved when Congress passed the Social Security Act. This act provided Americans with unemployment, disability, and pensions for old age.
The American Dream
Some may see the American dream as having a lot of wealth, big, fancy houses, and other "shiny" things.
Many people believe that the American Dream is one that "... emphasizes community, ecological sustainability, and a celebration of non-material values, while upholding the spirit of the traditional American dream of life, liberty, and the pursuit of happiness."
Others believe that the American Dream is to succeed in your career and to do what you love.
For example, in Of Mice and Men, Steinbeck uses themes such as friendship, loneliness, and being an outcast. To some people, this book might seem appealing to them if they relate to any of the themes.
John Steinbeck had used these themes to make his writing stand out and emphasize certain points that he was trying to show. Given the fact that his writing pieces are still read throughout the world today, I think putting all of his effort into his books was worth it.


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Perhaps this latest finding will help to stimulate the search for some more satisfying answers.
Astrophysicists will be scratching their heads following the discovery that three elliptical galaxies seem to contain little or no dark matter. A team led by Aaron Romanowsky of the University of Nottingham in the UK found that the dynamics of the elliptical galaxies could be explained without the need for dark matter, in contrast to the motion of spiral galaxies. The unexpected result questions the widely held belief that elliptical galaxies form when galaxies rich in dark matter collide.
Dark matter was originally proposed to explain why galaxies rotate as though they contain much more matter than astronomers can detect with telescopes. The existence of this invisible mass - and its gravitational pull on ordinary baryonic matter - has become a cornerstone of modern cosmology, but now Romanowsky and colleagues have cast doubt on its existence in certain galaxies.
For three elliptical galaxies, the researchers measured how the rotational speed changed from the centre of the galaxy to its outer edge. To do this, they used the 4.2-metre Herschel telescope in La Palma to look for planetary nebulas - shells of gas ejected by sun-like stars at the end of their lives. The spectra emitted by these shells contain well-known absorption lines, which are red-shifted for nebulas receding from Earth and blue-shifted for those approaching us. The degree of this Doppler shift enabled the researchers to calculate the velocities of hundreds of nebulas, and therefore their galactic neighbourhoods.
To their surprise, Romanowsky and colleagues found that the rotational speed of the galaxies fell towards their outer edges - a result that Johannes Kepler would have predicted long before the advent of dark matter. In contrast, the rotational speed of matter beyond the visible edge of spiral galaxies remains constant. Astronomers believe this arises from the gravitational effect of 'halos' of dark matter around spiral galaxies. Now they must explain why certain types of galaxy appear to be rich in dark matter while others seem to be deficient.


Even though Bethesda was not capable to deliver us a single piece of software that worked flawlessly (from the initial launch of Fallout 3 to the release of the latest DLC, Broken Steel), we still love the RPG and purchase the extra content. Apparently, that’s exactly what makes Bethesda wish to deliver even more of that. So yes, consider it a possibility!
Basically, the latest DLC, Broken Steel, removed the ending of the game and let it be, as the Beatles once said. In recent times, however, that means that, should the devs believe that they’ll have enough profits, new DLC packs will be released.
“Whether [Broken Steel is] the absolute last one remains to be seen,” said Bethesda’s Pete Hines to OXM. “Because it’s been hugely successful. Operation Anchorage vastly exceeded our expectation. It was like ‘holy s@*t, we did how many?’ and the Pitt was really successful, after a bit of a false start.”
The false start was caused by the problems with the DLC, of course. So I think that, should they manage to develop a pack that would come with no extra bugs, people would buy it. But I think they’re tired of getting not working DLC they have to wait for extra to play.


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Abu Dhabi non-oil trade up 15.7%
Asia represented the leading source of imports through the ports of Abu Dhabi with a share of 48.9 per cent in second quarter of 2015, compared with 26 per cent and 15 per cent for imports from Europe and North America, respectively.
Abu Dhabi - Non-oil imports rise 6.15% in Q2, exports and re-exports contribute 68.9%.
Abu Dhabi's non-oil merchandise trade rose 15.7 per cent year on year in the second quarter to Dh42.4 billion, said Statistics Centre-Abu Dhabi, or Scad, in its latest report.
The trade growth reflects an increase of Dh1.7 billion or 6.1 per cent year on year in non-oil imports, an increase of Dh4.4 billion or 97.5 per cent year on year in non-oil exports, offset partially by a decrease of Dh335 million in re-exports during in the April-June period of 2015.
Non-oil imports, exports and re-exports contributed 68.9 per cent, 21.1 per cent and 10 per cent, respectively, of total merchandise trade through Abu Dhabi Ports in the second quarter of 2015. According the report SCAD, 'machinery and transport equipment' was the largest contributor to imports, representing 50.8 per cent of the total. Its value increased by 10.7 per cent to Dh1.4 billion compared with the same period previous year.
The main contributors to this increase were 'other transport equipment', which advanced by Dh1.3 billion in the second quarter of the year. The report reveals an increase of Dh4.4 billion or 97.5 per cent year-on-year in the value of non-oil exports during the second quarter of 2015, with 'commodities and transactions' as the largest non-oil exports by Standard International Trade Classification comparison. The second largest SITC4 section was 'manufactured goods classified by material', although it fell by 10.6 per cent year-on-year, followed by 'chemicals and related products' that grew up 9.3 per cent year-on-year.
As SCAD's report finds, 'industrial supplies not elsewhere specified' was the largest category of imports representing 36.4 per cent of the total value, having grown by 5.2 per cent year-on-year to Dh522 million. The second largest category was 'capital goods', which increased 26.7 per cent, while the third largest was, 'transport equipment and parts', declined by 5.2 per cent to Dh364 million.
The combined contribution of these items was 87.8 per cent, in the quarter up against 86.1 per cent in second quarter 2014.The 6.1 per cent year on year rise in the value of imports was mainly due to an increase of Dh1.7 billion in 'ships, boats and barges' and Dh1.0 billion in 'pearls, precious metals, stones & articles'.
With a share of 48.9 per cent, Asia represented the leading source of imports through the ports of Abu Dhabi during Q2 2015, compared with 26.0 per cent and 15.0 per cent for imports from Europe and North America, respectively. The report reveals an increase of 97.5 per cent or Dh4.4 billion in the value of non-oil exports during the second quarter of 2015, with 'commodities and transactions not classified elsewhere', having increased by Dh4.3 billion in the period.
'Manufactured goods classified by material', however, fell by 10.6 per cent, while 'chemicals and related products' rose 9.3 per cent over the period under comparison. The 'industrial supplies' contributed 87 per cent to the exports during April-June period. This category showed an increase of Dh3.9 billion, up 100.1 per cent year-on-year.
The food and beverages, followed by consumer goods contributed 97.5 per cent of the total value of non-oil exports in the quarter in 2015, up against 92.7 per cent in the same period last year.
The rise in non-oil exports during the period was mainly due to an increase of Dh 4.4 billion in the exports of 'pearls, precious metals, stones and articles', partially offset by decreases of Dh257 million in the exports of 'aluminium and articles' and Dh248 million in the exports of 'copper and articles'.


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Hidden among us, alien machines with the awesome ability to change from one form to another fight their ancient battle for the AllSpark. They are. . .THE TRANSFORMERS! The DECEPTICONS have been beaten on Earth, but OPTIMUS PRIME knows they've got bases everywhere. After S7 shared their Mars data with him, he scanned his new camouflage and headed to the red planet hunting DECEPTICONS. Awesome quick-change figure converts from robot mode to flame-detailed tractor-trailer truck mode and back again! Robot mode features a launching "fire" blast!
Some of our vintage or prior versions of toy and game instructions may be more difficult to decipher or have less clarity. If you have any concerns or questions please contact our Consumer Care department at https://consumercare.hasbro.com/en-us


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Manchester United defender Victor Lindelof has been praised after tackling a thief who had stolen an elderly woman’s handbag.
Manchester United attacker Bruno Fernandes has played down an on-field argument with defender Victor Lindelof after seeing his side crash out of the Europa League.
Manchester United need to be taking a ‘ruthless’ approach to improving collective standards, says Gary Neville, with the Red Devils great looking for a centre half to be signed to compete with Victor Lindelof and clarity to be offered regarding David de Gea’s future.
Harry Maguire believes Manchester United must improve their record of set-piece defending.
Manchester United are sweating on the fitness of Marcus Rashford, Victor Lindelof and Harry Maguire ahead of this weekend’s trip to Bournemouth.
Manchester United reportedly plan to hand new contracts to trio Paul Pogba, Jesse Lingard and Mason Greenwood.
Manchester United have secured Victor Lindelof’s long-term future after agreeing to a deal with the defender until at least 2024.
Manchester United are reportedly ready to double Victor Lindelof’s wages with a new contract for the Sweden international.
Swedish international Victor Lindelof has reaffirmed his commitment to Manchester United after the defender’s agent raised doubts about his future.
Wolves sealed an FA Cup semi-final spot as goals from Raul Jimenez and Diogo Jota saw off Manchester United 2-1 at Molineux on Saturday evening.


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Book Critique - Masquerade
For reviews of other books in this anthology: Go here
Stars: 3 / 5
Recommendation: For those quick reads you are looking for in paranormal romance genre without any sequels or series to go through, this is a perfect book.
Masquerade is a 1994 novella by Amanda Ashley published as part of the anthology After Twilight along with novellas from two other authors - Christine Feehan and Ronda Thompson. This is the first of Amanda's novels with a vampire theme. It was originally published in the anthology The Topaz Man Presents Secrets of the Heart and later for After Twilight.
In the book credits on the first page, Amanda Ashley is credited as Madeline Baker. In fact Amanda Ashley is Madeline Baker's alter ego. While she wrote historical romances under Madeline Baker, she wrote books with vampire / paranormal theme under Amanda Ashley name. Quite a contrast in genre that she has for each of her names.
This first book, Masquerade, is a mere 112 page novella yet it did bring out her love for Vampires and the epic musical The Phantom of the Opera by Andrew Lloyd Webber. The current plot revolves around a 300 year old Vampire, Jason Blackthorne and his love towards a mortal, Leanne, who played a chorus role in the theater presentation of the famous Phantom of the Opera. A love that would not give any solace to either one yet they don’t deter to act on it. However how will the same love change their loves is what the rest of the plot is about.
It is very clear that Amanda (I am going to call the author Amanda as opposed to Madeline since that is what she wrote this book under) has definite affinity for the musical that she reiterates time and again in different scenes between Jason and Leanne. And also for Vampires. We see periodically that Jason and Leanne enjoy watching the famous Hollywood movies on vampires - the 1979 horror comedy film Love at First Bite, the 1979 British-American horror film Dracula and the 1931 American vampire-horror film Dracula.
A simple and sweet tale of love and sacrifice which I felt finished faster than it needed to be. I don’t believe she had written a sequel to this book, but she did continue writing more of vampire themed romance novels.
An easy and quick read that will entertain you just enough to finish it.
1) Grammatical / Geographical / Location / Character / Historical Errors:
a. On Pg. 68, line 13, there is no need for the "the" at the beginning of the line.
b. On Pg. 85, line 22, there is no need for the "the" at the beginning of the line.


Jumeirah Messilah Beach Hotel & Spa, Kuwait’s idyllic resort, has partnered with Porsche Centre Kuwait, Behbehani Motors Company to help raise awareness of environmentally friendly transport options.
The hotel will host the Porsche Panamera S E-Hybrid car at its main entrance to provide an alternative luxury mode of transport for the hotel’s VIP guests and celebrities, while at the same time raising awareness of hybrid cars and educating motorists about reducing fuel consumption and CO2 emissions.
The Porsche Panamera S E-Hybrid is the world’s first plug-in hybrid model on the luxury class market. The 416 hp model has an electric driving range of up to 36 kilometers after which the vehicle will switch to normal fuel or the driver can recharge it. The car has an NEDC fuel consumption of just 3.1 liters per 100 km which equates to CO2 emissions of just 71 g/km – the same level as a compact car. In addition to the Porsche Panamera S E-Hybrid, Porsche is a market-leader in the production of plug-in hybrid cars and is the only manufacturer to offer three hybrid models. Its E-Hybrid line-up includes the Panamera S E-Hybrid, 918 Spyder and the recently launched Cayenne S E-Hybrid – the world’s first plug-in hybrid in the premium SUV segment.
“This collaboration showcases our commitment to sustainability. We are proud to support an environmentally friendly initiative and to have partnered with Porsche – a globally recognised and respected automobile brand. It’s important to protect the environment against the harmful emissions of conventional fuel-powered, combustion engine cars. Our guests will not only enjoy the comfort of Jumeirah Messilah Beach Hotel and Spa but can now take advantage on environment-friendly luxury transport,” said Hakan Petek, General Manager, Jumeirah Messilah Beach Hotel and Spa.
Porsche Centre Kuwait, Behbehani Motors Company— Deputy Group General Manager Roger Kyriacou said: “For more than eight decades Porsche has continued to build on its unrivalled legacy of breaking new automotive ground in the field of sustainability. Porsche Centre, Kuwait Behbehani Motors Company has always been dedicated to environmental protection and finding innovative solutions to reducing fuel consumption and CO2 emissions.
The Porsche Panamera S E-Hybrid provides advancements in both efficiency and sportiness and is a pioneering vehicle because it delivers the dynamic performance and power expected from Porsche, while also helping individual owners to reduce their carbon footprint. We are proud of our partnership with Jumeirah Messilah Beach Hotel and Spa and the important work we will be doing together to raise environmental awareness.”
In its commitment to create a positive impact in Kuwait and engage its guests and colleagues, the Jumeirah Messilah Beach Hotel & Spa is actively involved in social responsibility initiatives and activities such as the Earth Hour, Breast Cancer Awareness, World Autism Day, student school projects, art competitions and beach clean-up drives.