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BEIJING – China’s economy recorded the first contraction in decades in the first quarter as the novel coronavirus that causes COVID-19 shut down large parts of the world’s second-largest economy and dimmed the global outlook.
Gross domestic product shrank 6.8% in the first quarter from a year earlier, missing the consensus forecast of a 6% drop and logging the worst performance since at least 1992 when official releases of quarterly GDP started. Factory output fell 1.1% in March, retail sales slid 15.8% and investment decreased 16.1% in the first three months of the year.
Policymakers will nevertheless take encouragement from the marked improvement in industrial output, which had suffered a double-digit fall in the first two months. A key question for recovery prospects is the extent to which consumers regain confidence as the lockdowns are lifted.
"The data confirm China is, for now, healing,” said Shaun Roache, APAC Chief Economist at S&P Global Ratings. "Still, it is becoming clear that consumption will lag as the economy enters the transition period between lockdown and vaccine.”
That could reinforce the need to keep stimulus settings moderate for now, especially as the spread of the virus around the world also threatens to add fresh downward pressure on China’s exporters in the months ahead.
China’s markets held gains after the release as investors digested the data. The Shanghai Composite Index was up 0.8% at 10:38 a.m., while the Hang Seng Index climbed 2.6% in Hong Kong.
China’s economy was forced into a paralysis in late January as the virus first identified in Wuhan spread across the country. The economy remained shuttered for much of February, with factories and shops closed and workers stranded at home. The process of resuming business has been disappointingly slow and the return rate only inched up to around 90% at the end of March, Bloomberg Economics estimates.
To cushion the economic blow China has unveiled a range of support measures, although not on the scale of other nations. That includes 3.55 trillion yuan ($502 billion) in low-cost funding provided to financial institutions, 1.29 trillion yuan in preapproved local government special bonds and 1.6 trillion yuan in cuts to various fee taxes, according to the nation’s cabinet.
The central government is also mulling other policies like raising the deficit-to-GDP ratio, issuing special sovereign bonds and increasing the local government special bond quota, in order to fuel a faster economic recovery, according to a recent article from a senior official.
Exports fell less than expected in March as production capacity was gradually restored. But economists warn bigger headwinds lie head as the rest of the world shuts down and external demand diminishes.
"Most major economies are still in the lockdown stage,” said Robin Xing, chief China economist at Morgan Stanley Asia, on Bloomberg TV. "As a result growth in the second quarter will be shallow, just marginally above zero.”
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on 28 January 2013
The three movies to thus far feature Daniel Craig as James Bond 007 are collected here as a DVD triple bill. And as many will already know, it's certainly a case of `two out of three isn't bad'...
2006's Casino Royale was the finest James Bond movie in years; in this film Craig gave us (and I know this is a cliché) arguably the most striking Bond since Sean Connery's early days, erasing the cheesy pong of Pierce Brosnan's tenure as 007 and investing the character with an emotional depth not seen since On Her Majesty's Secret Service back in 1969; and unlike the stiff George Lazenby, Craig is an actor with the skill to pull off his more dramatic scenes convincingly. Just about everything else about the movie hit the bullseye too; it certainly deserves a score of 8 out of 10, only losing a few minor points for the bland theme song, the opening credits sequence (which was a bit too `Casino Royale 1967' for my taste), the last-minute `excuse' for Vesper Lynd's treachery, and of course Judi Dench's continuing on in her role as the `headmistress' M; she may have a lot of support from mainstream critics and was clearly regarded as an asset by the producers, but here, her presence is an unnecessary holdover from the Brosnan movies, and the one jarringly anachronistic note in this `Year One' outing.
Following on directly from the final scene of Casino Royale, Craig and Bond returned in 2008's Quantum of Solace, a very unsatisfying movie that retains the style and feel of its forerunner but carries none of its emotional weight or resonance. The plot of this direct sequel is weak, contrived, and almost incomprehensible. The musical score is dire, the action scenes (whilst competent) lack logic, the villains are useless (a stereotypically sleazy, corrupt Latin American General, and Mathieu Amalric's sub-Blofeld `mastermind', who is so ineffectual it is impossible to believe he could be a big wheel in the organisation that Casino Royale's Le Chiffre was so afraid of), and the Bond girls are dull and uninteresting. Craig is clearly trying to show how the character of Bond is growing `harder' as he gets older, but instead comes across merely as a swaggering, pouting, humourless bully, whilst Dench is yet again given far too much to do (`I'm going to send you out on your own into hostile territory 007...but I'll still show up with a gang of agents in tow every ten minutes to stick my oar in'). With fudged revenge themes, pointless deaths of characters we are meant to care about (but don't), a silly climax, and a final scene that tries desperately to copy the chilly sang-froid of the ending to The Bourne Supremacy, and fails spectacularly, one's reaction to Quantum of Solace as the credits roll is `what was all that about?'.
Thankfully, 2012's Skyfall was a fine return to form. Clearly conceived as a genuine 50th Anniversary tribute to Bond's status as a pop culture icon, the film manages to homage just about every other movie in the series as well as delivering a hard-hitting thriller plot that updates (and arguably re-boots) the 007 formula. There is little to say about the film other than that it is lovingly crafted in every area, features a witty and emotionally resonant script, and features Craig's best movie performance thus far, not to mention having, in Adele's contribution, the very best Bond theme song since Carly Simon's `Nobody Does It Better' featured in 1977's The Spy Who Loved Me. And if it received perhaps a little too much praise from critics (especially with regard to the contributions of Dench and of Javier Bardem as the villain), whilst the essentially derivative nature of its storyline was conveniently ignored (despite being considerably better than either film, the plot is essentially a mish-mash of elements from The Man with the Golden Gun and The World Is Not Enough), the only really distressing thing about it is that, like The Dark Knight Rises, it follows up two movies charting their iconic hero's `early days' with one that fast-forwards straight through to the `autumn' of his career; but unlike Christian Bale's Batman, there is little doubt that we haven't yet seen the last of Craig's James Bond...
As one of the only 12 people who was genuinely delighted at Daniel Craig's casting when it was announced , I must admit I was more than a little worried about Casino Royale. Not the kind of paranoia that those newcomers who'd never experience the changing of the guard the series goes through every decade or the staggeringly venomous hate-mongering of the more fanatical Brosnan fans who felt compelled to start libellous hate-sites, though. After all those months of arguing that he was the perfect choice for the role (especially after some of the more moronic suggestions), was I setting myself up for a fall if he turned in a disappointing performance? Similarly there was the film itself. While the producers were making all the right noises about going back to basics, they'd done exactly the same with Licence To Kill and chickened out to deliver a sub-Roger Moore effort with Wayne Newton as a comedy relief villain, inept ninjas, pointless gadgets, laughable violence and monster truck stunts. Too often in the past the franchise had been over-reliant on the goodwill generated by the earlier films, rehashing earlier vehicles to decreasing returns secure in the knowledge that the audience would turn up anyway. Take away the Bond brand, and too many post-OHMSS entries simply wouldn't have stood up to scrutiny in the marketplace on their own merits: Bond had become a tradition, a ritual like going home for the holidays that you knew was never going to be as good as it was when you were a kid but which you still went through out of a mixture of hope and obligation.
I needn't have worried. Not only is it the best Bond film in 37 years, it's as if the Roger Moore and Pierce Brosnan years never happened. After Brosnan's surprisingly lazy and slightly seedy turn in Die Another Day, Craig delivers the most physical Bond since Lazenby, but this time matched by the acting chops to make the most of the best script the series has had in decades - at once plot and character led - as the rookie blunt instrument bulldozes his way through his mission until emotional awakening and betrayal starts to finesse him into the Bond we knew from the Connery days. Brosnan never could have delivered this kind of performance, either physically or emotionally, and, truth be told, neither could Connery in his prime: Craig is the first one to convince you that he's not a movie star or an actor but that he really IS James Bond.
The updating of the plot from the Cold War era to a post 9/11 world works surprisingly well, with the first act managing to provide a convincing motive for the high stakes poker game centrepiece while also providing a couple of superb action scenes that don't become too absurd and serve the plot in a series where in the past the plot was too often an excuse for the action. The much-criticised change from baccarat to poker is a smart one too. Where Baccarat is purely a game of luck (as Fleming himself found out when he went bust in three hands trying out the novel's premise on a Nazi spy), poker actually involves both strategy and psychology, making for more satisfying drama and tension.
There is, sadly, one concession to gadgetry that veers into the absurd - c'mon, who keeps a defibrillator in their glove compartment? - and is an unwelcome reminder of the days when old Roge would get out of a scrape with his buzz saw wristwatch or his projectile dart cufflinks thanks to lazy writing, but elsewhere it settles for using existing technology (most of it manufactured by Sony for some reason that escapes me) rather than veering into total fantasy. And it's good to see a Bond who needs hospitalisation after the villain goes Quasimodo on his nuts with a bell rope. The real James Bond is indeed back.
As per the edition included in the 50th anniversary Blu-ray boxed set, the version of Casino Royale in this triple pack is a strange hybrid of the two-disc special edition and first single-disc release, containing most of the special edition's extras but losing Martin Campbell's picture-in-picture commentary, two featurettes (The Art of the Freerun, Catching a Plane - From Storyboard to Screen), the revised documentary Bond Girls Are Forever, storyboard sequence and filmmaker profile featurettes (Martin Campbell, Chris Corbauld, Phil Méheux, Gary Powell, Alexander Witt and David Arnold).
I'll admit that I went into Quantum of Solace more or less dreading a repeat of the Licence To Kill debacle. All the danger signs were there - a rushed script because of a writers' strike, threats of Bond going rogue again plus the problem that great Bond films are usually followed by naff ones. The short running time wasn't encouraging, nor the bigger budget and promise of more action.
Well, this isn't one of the great Bond films, and Casino Royale set the bar far too high for it to compete. But it's certainly not a disappointment if you go in aware of that, and more gratifyingly, the similarities to Licence To Kill are superficial. Where Casino Royale was like making love all night long, this is more of a gratifyingly frenzied *beep* of a film. The running time isn't a problem because, like From Russia With Love, this is a pared down machine with no fat to trim away, throwing out all the overused touchstones to get down to business. From a plot point of view there's maybe a little too much one corpse leading to the next plot point in the first third, but the film wisely ditches that approach early.
Dan Bradley's action scenes are thankfully not as ineptly over-edited and incoherent as in Paul Greengrass' films, but aren't as impressive as Gary Powell's work on Casino Royale. There are moments of familiarity - a motorbike sequence borrows from the unimpressively shot harbour scene in Jackie Chan's The Protector, but without the lethargic pacing, while an aeriel dogfight owes a lot to a famously rejected stunt originally intended for the opening of GoldenEye - and the opening car chase through heavy traffic could have benefited from not trying quite so hard. But within them there are moments of stylisation that few other Bonds have attempted and failed at but which are far more successful here, most notably an impressive opera sequence that could have done with a few more shots to clarify the odd mechanical detail (something other parts of the film could benefit from). It's also surprisingly vicious - for perhaps the first time in a Bond film, innocent bystanders are deliberately killed. That said, the rationale for the explosions at the end is more than a little dubious.
The film isn't as humorless as some have complained: there's a lot of dry humor where appropriate and a delightfully playful game of cat-and-mouse with Bond and M in a hotel, but none of the outright slapstick comedy that dragged the series down before. Nor is Forster's direction or the editing as awkward as some found it: there's a pleasingly epic scale to the film allied with a non-nonsense straight-down-to-business attitude that works well for this particular story.
The most curious complaint is that it's just action with no character development, when nothing could be further from the truth. While there is more action, the characterisation is integrated into both plot and action. Bond is once again on an emotional journey - forgiveness, believe it or not, is ultimately the quantum of solace of the title - though this time the heart and soul of the film is Giancarlo Giannini's Rene Mathis, the kind of man Bond might be capable of becoming and one he learns something about himself from. One of their scenes is easily one of the very finest moments in the entire history of the series.
Craig still owns the role impressively and Jeffrey Wright starts to come in to his own as Felix Leiter this time round. Mathieu Amalric is one of the better villains of the past twenty years. He won't be among the greats, but he convinces and the scheme is genuinely ingenious in its simplicity. Olga Kurylenko manages to shake off the ineptness of her former performances to be a more than adequate but not especially memorable female lead, though Gemma Arterton lets the side down badly in a part that has unwelcome elements of Serena Gordon in GoldenEye and Rowan Atkinson in Never Say Never Again. Thankfully it's a small role so her weak and stilted straight-out-of-stage-school acting can't do too much damage.
Intriguingly, the film exists in a more convincing world of global politics than we've seen before in a Bond film: SPECTRE would have loved to be around in an era when governments eagerly step into bed with crime syndicates if it suits their ends and where corporations are able to play governments and intelligence agencies against each other. Here Bond works for a British government that tortures suspects on foreign soil and blindly goes along with foreign interests and crime syndicates alike in its desperation to snatch the scraps from the superpowers' tables. Initially, Bond is just as ruthless and morally flawed as his masters, the bullish arrogance gradually being smoothed away by emotional experience as he learns the importance of forgiveness to find the quantum of solace of the title that he needs to go on.
Yes, there are weaknesses - M's office is overdesigned, a few scenes could have played better with more time, the Goldfinger reference is unnecessary, the song is crap and the gunbarrel sequence is a big and unnecessary mistake - but it's not the crushing disappointment some are claiming. It may not be a great Bond movie, but it most definitely is a Bond movie, and a damn good night out at the pictures. And one that left me seriously thinking that even if the series never recaptures the high of Casino Royale,we may just be entering a genuine second golden age of Bond movies.
The extras package isn't quite as bad as some make out, but it still seems like a stopgap until the inevitable special edition set comes out shortly before some future Bond film makes its bow. There's certainly nothing to get too excited about - the half-hour On Location featurette aired on TV at least three times last winter, the 43-minutes of brief crew interviews were on the net while the five short featurettes fee like electronic press kit packages. The terrible music video for the terrible title song and two trailers for the film are also included.
The Craig Bond films manage the surprising feat of being true to both the novels' and the movies' legacies, and at the heart of John Logan's script for Skyfall is a good idea (apparently a leftover from Peter Morgan's abandoned treatment that was put on hold when MGM/UA went into bankruptcy, delaying the film by two years): everything we associate with Bond and his past is reintroduced and taken from him or destroyed before the classic Bond is resurrected in a way that's true to both the novels and the films. Where Casino Royale went back to the very beginning, Skyfall uses a lot of both the darker plot of the novel The Man with the Golden Gun (a washed out Bond returns from the dead and is sent on a mission that will either redeem him or conveniently see him killed off) and the siege finale of Fleming's (rather than EON's) Spy Who Loved Me and reworks them for the present day.
In many ways the film is a journey into the past, starting out as an epic globe-trotting adventure before scaling down to Bond's childhood Scottish roots and finally bringing the series full circle with Moneypenny back in the same blue dress she wore in Dr No and M no longer in a hi-tech office but the old wood-panelled one with the same padded door and the same naval painting on the wall from the Bernard Lee days, rewinding the series to the Connery era. If hats were still in fashion, Craig would have made his entrance throwing his onto the hat stand. It's a deliciously understated scene that goes uncommented on - thankfully the film never feels the need to knowingly wink at the audience - that will have Bond fans of old feeling like a puppy dog with two tails. And the new faces as the old regulars (Ralph Fiennes, Naomie Harris, Ben Whishaw) may be very different from their predecessors, but they fit very nicely with this new/old Bond.
Unfortunately, on the minor debit side Logan has always had a bit of a problem with his villains. He can do plot or milieu or character, but he can't really pull off any combination of the three with his bad guys, and while this is a bit of an improvement he does fall back on the tried and trusted. In Gladiator he offered a mad lisping Emperor with a chip on his shoulder betraying the people who put him in a position of trust to pursue a vendetta with a brother figure. In Star Trek: Nemesis he offered a petulant lisping mastermind with a chip on his shoulder betraying the people who put him in a position of trust to pursue a vendetta with a father figure. In Skyfall he offers us a devious lisping mastermind with a chip on his shoulder betraying the people who put him in a position of trust to pursue a vendetta with a mother figure. All he needs now is someone with a lisp pursuing a vendetta with a sister figure and he'll have the full set of immediate family adversaries.
On the plus side, this villain is intelligent and actually has a masterplan instead of just talking about one, even if it is one that at times suggests he's a bit of a fan of The Dark Knight, and he's played by a much better actor, though that is something of a double-edged sword. Despite being given a great build up - the slight physical trembling and almost-disguised weakening of the voice Severine gets even thinking about him is a wonderfully underplayed appetiser - he's a rather thin character, leaving Javier Bardem trying too hard to make a memorable impression with the kind of quirks that great actors who are persuaded to make more commercial fodder than they're at home in throw into the mix to keep themselves from getting bored (think Brando on his off days).
It's a bit of a letdown considering what Bardem is capable of, but for some reason every time an Oscar winning actor who can do menace gets a sniff of a Bond villain they dye their hair blonde and camp it up. Thankfully he doesn't go the full John Inman, but the film is strong enough not to need the theatricality or the absurdly bit of obvious CGI thrown in to his Hannibal Lector scene. While he doesn't ruin the film, I think the performance is why the film hasn't grabbed my affections the way Quantum did even though they improved the problem areas from that: I found Mathieu Almaric a far more intriguing villain because he wasn't playing the villain, he was playing the good guy (or at least ecologically aware businessman), and his scheme less reliant on an almost supernatural ability to predict the good guys' every move years in advance. And while I liked the exploration of M's ruthlessness, her departure didn't have the same impact as that of Mathis in Quantum.
While it's pleasing to have Stuart Baird, quite possibly the greatest editor of action scenes alive today, back in the editing suite after the often misjudged editing in Quantum it's a shame that composer David Arnold was replaced with Thomas Newman, who astonishingly became the first in the series' history to be nominated for best score despite a truly anonymous temp track-style score with no personality, themes or development (although it works much better on the film than as an album where it's derivative general shapelessness is much more exposed).
Curiously the Blu-ray exaggerates what were many of the niggles about Roger Deakins' lazily overvalued cinematography (you do get the impression that had the identical images been created by a cinematographer who wasn't a name it would have attracted little attention), with many scenes like the Board of Inquiry or London street scenes suffering from the lack of depth, slightly bleached look and waxy loss of detail that are among the most commonplace drawbacks when you don't shoot on film. It's strange that Deakins got a free pass on this one when the move from film to digital has had such inconsistent results both in terms of quality and creativity: it's a mixture of a few strikingly good looking scenes (the Chris Doyle/Blade Runnerish fight against a neon background) and a lot of very drab or visually clichéd ones (seriously, that relentlessly dreary rainy London look has been done to death for years by television) while there are some real problems with focus and colour balance in the opening to the Macau casino scene and parts of the siege suffer badly from digital's ongoing inability to handle shadows and low light levels very well. It's a long way from his best work, and to claim, as some have done, that it's the best looking Bond film is doing a great disservice to the work of Freddie Young and Ted Moore on earlier, much better looking films. It doesn't help that there's some very noticeable edge enhancement in places - the doorframe to M's office and his picture frames look like a straining rope or teeming with termites from one frame to the next.
Yet while its flaws keep it from reaching the heights of Casino Royale, there's so much to enjoy and so much it gets right and strikes such a good balance between exciting action scenes and a real look at what makes Bond Bond that it's still a worthy addition to the franchise - not top tier, but certainly no middling effort either.
The extras package looks substantial - two audio commentaries, some 15 featurettes and trailer - but at times there's more puff than substance.
on 1 January 2013
Daniel Craig has one facial expression, many guns and women and a reasonably popular stance as James Bond. He is very much the closest to the Ian Flemming character of the books, a cold, heartless and efficient killer with women problems, gambling addiction and other flaws. He's not so much the dashing bravado with the punchlines but he does resonate well in the context of his character's work. The three Bond movies he's been in (2005-2012) vary in quality, one is great, one is good and one is awful / shaky. Not in that order mind you.
Casino Royale is a very good start and the film which rejuvenated Bond in the 21st century. It's a glamorous, gritty story with not much in terms of gadgets, plenty of women and fights, as well as Bond being rammed in the nuts several times with a chain. It's the good one, stylistic, good use of locations, relatively interesting characters and it builds up nicely to a suitable climax. The film takes a kind of origin stance in that James is new to the game, a brutal loose cannon just elevated to 00' status. The movie is essentially good and showed promise for both Craig and the Bond movies he would follow.
Then there's Quantum of Solace. Taking perhaps too many lessons in camera work from the Bourne movies this s basically a low stakes, incoherent mess of a movie where the camera shakes a lot, the conflict is low key and there's not a single ounce of charm. Now on one hand this works as it follows immediately from the tragedy of Casino Royale and the gritty, angry nature of Bond is quite an effective drama source. But it's not well done, not well filmed and the movie is bland. It lacks charm, visual interest, camera steadiness or interest. Water control is what we're fighting over when vengeance is off the table and being honest, it's not quite James Bond level of stakes. So overall, Bond looked quite sunk with this one, a bad, bland movie.
The best of the three however is Skyfall. Skyfall is engaging on all levels, its got charm, its exotic, it has good engaging characters, a great villain with great lines, an engaging and highly dramatic conflict, a sense of fulfillment and satisfaction, hints at the past of our elusive protagonist and the action is some of Bond's best. Combining engaging performances from all (OK Craig's one facial expression and lack of interest is something of a problem here but it somehow balances against everything else as our anti-hero makes this journey). There's actual spying going on here, looks great, script is solid and overall, for me, this is the best Bond film, engaging and entertaining with the safety barriers broken in minutes and the stakes higher than ever on a personal level.


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In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.
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Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Institutions: Georgetown University Medical Center.
Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo
. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo
. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-κB (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-α (TNF-α), upon lipopolysaccharide (LPS) and Pam3
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Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.
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replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity
Institutions: New York Medical College.
the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.
Immunology, Issue 97, Toxoplasma, macrophages, innate immunity, intracellular pathogen, immune evasion, infectious disease, forward genetics, parasite
Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster
Institutions: Cornell University.
The fruit fly Drosophila melanogaster
is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila
can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.
Immunology, Issue 99, Drosophila, immunity, infection, resistance, tolerance, bacteria, Providencia, antimicrobial peptides, immune defense
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Institutions: Institut du cancer de Montréal, Université de Montréal.
During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer’s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently gene expression was evaluated.
Immunology, Issue 98, Peritoneal cavity, macrophages, thioglycollate, infection, inflammation, TLRs, RNA extraction
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs
-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf
transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf
animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1
. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf
fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Institutions: The University of Texas at Austin, The University of Texas at Austin, The University of Texas at Austin.
Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.
Chemistry, Issue 79, Membrane Lipids, Toll-Like Receptors, Endotoxins, Glycolipids, Lipopolysaccharides, Lipid A, Microbiology, Lipids, lipid A, Bligh-Dyer, thin layer chromatography (TLC), lipopolysaccharide, mass spectrometry, Collision Induced Dissociation (CID), Photodissociation (PD)
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes
Institutions: Johns Hopkins University.
In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
Cellular Biology, Issue 5, Translational Research, mosquito, malaria, virus, dengue, genetics, injection, RNAi, transgenesis, transgenic
Pyrosequencing: A Simple Method for Accurate Genotyping
Institutions: Washington University in St. Louis.
Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.
Cellular Biology, Issue 11, Springer Protocols, Pyrosequencing, genotype, polymorphism, SNP, pharmacogenetics, pharmacogenomics, PCR
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Institutions: University of Oxford.
The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3
. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4
. The primer extension assay is carried out using the MassARRAY (Sequenom)5
platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.
Cellular Biology, Issue 45, Gene expression, regulatory variant, haplotype, association study, primer extension, MALDI-TOF mass spectrometry, single nucleotide polymorphism, allele-specific
In vitro Biofilm Formation in an 8-well Chamber Slide
Institutions: The Research Institute at Nationwide Children's Hospital.
The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.
Infectious Disease, Issue 47, confocal microscopy, therapeutic approaches, chamber slide
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Institutions: Mount Sinai School of Medicine .
Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1
. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3
. Thus, DCs link the innate and adaptive immune systems.
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8
. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12
. Electroporation has been used with mixed results13,14,15
, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16
. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNβ
. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17
, at both the mRNA and protein levels.
Immunology, Issue 53, Dendritic cells, nucleofection, high-throughput, siRNA, interferon signaling
Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
Institutions: Université de la Méditerranée.
RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans
, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1
. While the creation of libraries of RNAi clones covering most of the C. elegans
opened the way for true functional genomic studies (see for example 4-7
), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8
. The approach relies on microscopic imaging and image analysis.
Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans
, since certain microbes do not efficiently infect worms in liquid culture.
We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans
. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29
and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9
. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29
expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.
Molecular Biology, Issue 60, C. elegans, fluorescent reporter, Biosort, LIMS, innate immunity, Drechmeria coniospora
Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood
Institutions: Yale University School of Medicine .
Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2
. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3
. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4
and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5
. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-κB, which initiates transcription of numerous genes that are critical for immune responses8
. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-κB pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9
Immunology, Issue 62, monocyte, dendritic cells, Toll-like receptors, fluorescent imaging, signaling, FACS, aging
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Institutions: Massachusetts General Hospital.
We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla
luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla
luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.
Cellular Biology, Issue 88, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Robotics
Establishment and Characterization of UTI and CAUTI in a Mouse Model
Institutions: Washington University School of Medicine.
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli
(UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis
. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis
bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Medicine, Issue 100, Escherichia coli, UPEC, Enterococcus faecalis, uropathogenic, catheter, urinary tract infection, IBC, chronic cystitis

domain: medical


Sharing photos from a shoot, Alia Bhatt slayed in a checkered mini dress which came with a dramatic with a cape. The ‘Brahmastra’ actress finished off her OTTD with stylish waist belt and a low ponytail. On the other hand, Shraddha Kapoor looked equally trendy in her checkboard outfit which had a stylish bow detailing on the shoulder. With a choker-style neck detail, the one shoulder bodycon dress is all things gorgeous.
On the work front, Shraddha Kapoor will be next seen in ‘Saina’, which is a biopic on badminton ace Saina Nehwal. While Alia Bhatt will be seen essaying the lead in sci-fi drama ‘Brahmastra’, wherein she will romance beau Ranbir Kapoor.


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This is a good one. My wife hates to see these sausage hanging at the side of the road. She always comments ” I wonder how long they have been there”. She has a point!
Anyways looks a good picture.
Fore All Golfer!
Golf, Courses, Equipment and Lots More
Life stories, road adventures, and big dreams by Marron Santillan
Until I know this sure uncertainty, I'll entertain the offered fallacy.
love each other like you are the lyric to their music
Tell Story Throughout the Images
Daily inspiration in decor style creativity
Avid adventurer, travel blogger, and experience seeker. Starting each morning with a desire to see the world through a different lens.
News, Opinion and Poetry
Photographs, Video and Stuff
nomadic, poetic, and sometimes ascetic
Fashion, Family, Food... Life!
Scribbles of an earthling somewhere 😊😊
The world around through my camera's lens
... and met these people on her way

domain: hobby


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Attorney firms in Hout Bay and surrounds
1 - 2 of 2 results
Henkes Joubert Barry Inc
"That is sufficiently certain that can be made certain"
Shop 15 Mainstream Mall Hout Bay
Miltons Matsemela - Houtbay
We offer experienced attorneys and conveyancers to assist with all property related transactions.
Unit 8, Medical Centre, Victoria Avenue, Hout Bay.

domain: law


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www.amazon.com This kit consists of a light-resistance, four-foot-long, six-inch-wide body band for sculpting arms, legs, and torso according to Pilates principles and a 55-minute workout DVD featuring well-known Pilates instructor Ana Cabán.
Video Rating: 5 / 5
www.PilatesFreestyler.com Pilates Freestyler™ Functional Dynamics intro. Discover how this new Pilates Reformer can make you enhance yor Pilates workout
Video Rating: 0 / 5


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NHK just announced a games TV show called Game Genome where people will be able to talk and play games for a full hour and they will be able to do so with the director / producer of the game as a guest.
For the very first episode, the TV show will have a very special guest to commemorate it by starring Death Stranding director Hideo Kojima.
The show will present the history of the game and the full âgenomeâ of the gaming community and industry. They will also present tips on how to play the games, much like taking a deep dive into the games in some way. so.
The show will also touch on the culture of the game and why it is fascinating. The show will be aimed primarily at gamers and those who unfortunately had to hang up their controllers. One of the goals of the show is to let them learn more about the games they love. The show can be enjoyed alone or with friends or family.
The show will be hosted by Tsubasa Honda, a popular Japanese actress known to be a Monster Hunter player and veteran.
Kojima had this to say about the show (according to our translator): “In my 35 years in the gaming industry, I’ve never been happier to introduce a game like this to a show. . In fact, I want them to make me a regular guest this time around! People who think like “Games are bad for you.”, I think I want them to watch this show. I’m sure that they will understand how much fun games are if they watch this show. I’ll be happy as long as people love games when they watch this show, like “I wanna try dancing like this!”. “
The show will begin airing on October 15 at 10:45 p.m. JPN time and will run for an average of 30 minutes.
Source: NHK Famitsu interview


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How Cornerstone Partners are improving prospects for underrepresented founders
|Date:||18.09.2020 - 18.09.2020|
Rodney Appiah, co-founder, and CEO of Cornerstone partners is joining our Friday Focus webinar on 18th Sept to share his initiatives for increasing funding for founders in underrepresented backgrounds.
Rodney will discuss how they are enabling more black and diverse founders to scale their business & grow their network through their Cornerstone 2.0 programme as well as give us an insight into research they have done alongside Diversity VC and Beauhurst to get a better understanding of diversity in UK VC’s.
Meet the speakers:
Rodney is co-founder and Chairman of Cornerstone Partners, a leading angel network focused on black and diverse founders in the UK. Rodney has worked in venture capital for nearly a decade and has been involved in six exits during that
Rodney is a Director at Foresight Group, one of the UK’s leading and longest established investors in growing businesses, and was formerly a senior investor at the Business Growth Fund (BGF), one of the most active growth capital investment firms in the world. Prior to BGF, Rodney began his career at Merrill Lynch where, over a five year period, he closed more than 15 transactions ranging from £50m to £1.2 billion in size. A former school governor, Rodney currently sits on four UK boards, mentors diverse entrepreneurs and aspiring VC investors, and is chair of the CBI London Under 35 committee.

domain: news


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This Cabin Airbnb In Mississippi Comes With Its Own Lake
By Lisa Sammons|Published December 28, 2021
Lisa loves animals and has dogs, rats, cats, guinea pigs, and snakes. She is passionate about animal rescue and live music - traveling across the country to see a favorite band is a pretty regular occurrence! Being out hiking in the woods enjoying the scenery with her beloved dogs is another favorite hobby, and also checking out the Pokemon Go scene in whatever city she happens to be in at any given time (coffee and dog leash in hand). You can reach Lisa at firstname.lastname@example.org
As local travel experts, we know what travelers are looking for when it comes to finding the perfect accommodations for their next trip. To compile our lists, we scour the internet to find properties with excellent ratings and reviews, desirable amenities, nearby attractions, and that something special that makes a destination worthy of traveling for.
When it comes to overnight stays in Mississippi, there’s a lot to choose from. You can get in touch with nature by pitching a tent in one of our 24 state parks. Or, if you dare, you can check out one of our notoriously haunted inns. Not up for the rigors of camping? Glamping’s always an option! With so many choices, it takes a lot to stand above the crowd. We think we’ve found one gorgeous Airbnb that takes the cake. How perfect does a cabin with a private lake sound?


text: Discover releases, reviews, credits, songs, and more about Cocteau Twins - Blue Bell Knoll at Discogs. All tracks from Bluebeard were included in the 2018 release Treasure Hiding: The Fontana Years. 1988 CapitolHere, by popular demand, is a complete discography of the Cocteau Twins' releases, not counting bootlegs. Les Cocteau Twins commencent leur carrière en 1979, et se séparent en 1997. The group was founded in the Scottish town of Grangemouth in 1981, when Robin Guthrie (guitar, production) and Will Heggie (bass) met Elizabeth Fraser in the town's disco. Sadly no longer with us but the music will live on and hopefully future generations will discover and fall in love with them and come to recognise that Elizabeth Fraser was possibly the greatest voice ever committed to vinyl. They are pure magic. Show more Written by Cocteau Twins. Biography The Cocteau Twins were a UK post-punk band, named after an early and obscure Simple Minds song. Liz makes you dream, and music seems to get married with the best female voice. Complete your Cocteau Twins collection. The group signed to 4AD Records and planned to record a two-track single. Find album reviews, stream songs, credits and award information for Treasure - Cocteau Twins on AllMusic - 1984 - The opening two numbers of Treasure are simply… A group whose distinctly ethereal and gossamer sound virtually defined the enigmatic image of the record label 4AD, Cocteau Twins were founded in Grangemouth, Scotland, in 1979. For anyone new to CT browsing the albums on this page, be sure not to overlook The Moon and the Melodies. Their best run, better than the albums for me, all sound great and look great to boot. Elizabeth Fraser does not use any specific language to write lyrics, Unfortunately, I discovered the Cocteau Twins late, but not a day goes by that I feel like listening to Liz, unfortunately many people don't know the CTs, and I'm sorry for them. Everything here is available from 4AD Records (London). They are pure magic. Find Cocteau Twins bio, music, credits, awards, & streaming links on AllMusic - One of the most distinctive bands of the '80s,… Liz has a voice that'll die with me, and send me off This Mortal Coil. I thank God for John Peel for introducing them to me during his shows. Someone did badly, I had to be born at least 7 years before, and not to bomb Metal, but to know all these bands in their best years. Grab all them mid period 12"s from Spangle Maker through Love's Easy Tears for maximum loveliness. When you hear them for the first time, you immediately fall in love with them, and the lyrics seem to be made on purpose, to let you enter another dimension. No other band has come close to being a true original in the way the Cocteau Twins did, with a sound and style that came from nowhere and was unique to them. They're not supposed to be interpreted!!! Put on a good pair of headphones, close the curtains, maybe light a few candles, lie down somewhere comfortable, and be prepared to drift off into another universe. I never really thought about it until I saw this listing - they really didn't put out that many albums - most were EP's. Find Cocteau Twins discography, albums and singles on AllMusic AllMusic. Mixed at Blackwing Studios, London, engineered by John Fryer. What gets my goat is people interpreting Cocteau twins lyrics into English. The group signed to 4AD Records and planned to record a two-track single. A guide to Cocteau Twins: biography, discography, reviews, links. They were formed in Grangemouth by Robin Guthrie (guitars, drum machine) and Will Heggie (bass), adding Elizabeth Fraser (vocals) in 1981 and replacing Heggie with multi-instrumentalist Simon Raymonde in 1983. This discography catalogs the first editions of these recordings as released … Cocteau Twins were a Scottish gothic rock and dream pop band whose discography contains nine studio albums, seven compilations, and numerous EPs and singles. Only "The Pink Opaque" and the last two albums have been released in the US, but any competent US record store should carry most of the stuff listed here. In the years since, several additional collections and special editions have been released by various record labels. The Cocteau Twins were a UK post-punk band, named after an early and obscure Simple Minds song. Too bad, but for about twenty abundant years, I discovered the 4AD, and I'm enjoying the various sounds of those years, discovering groups that excite me in hearing them and the CTs are the best, but the others are no less, Ivo is a genius who put under his label, the biggest bands, that music has ever produced, A huge influence on me, growing up as a teenager throughout the mid 80's, and I suppose, up until now. Seeing them live in the Sadler's Wells in London was. Cocteau Twins were a Scottish dream pop band active from 1979 to 1997. Guide D'Utilisation de la Base De Données, https://www.discogs.com/Harold-Budd-Elizabeth-Fraser-Robin-Guthrie-Simon-Raymonde-The-Moon-And-The-Melodies/master/4496, https://musictales.club/article/emotion-over-meaning-enigmatic-lyrics-cocteau-twins-languages-glossolalia, Great music, Weird Music, Important Music, The Great Rock Discography Survey: Martin C Strong's Best Artists And Songs In 1995, SƘƩLƩTÖƝ ƘİSS ƦɅĐIØ ' ' ' women of deathrock, Meat Beat Manifesto Satyricon Sleeve Notes. “Bluebeard” has appeared in live performances, and was performed live on “The Tonight Show with Jay Leno”—Cocteau Twins’ first television appearance in the US. Explorez les références de Cocteau Twins sur Discogs. … Unfortunately, I discovered the Cocteau Twins late, but not a day goes by that I feel like listening to Liz, unfortunately many people don't know the CTs, and I'm sorry for them. New Releases. Le style du groupe se caractérise par des tendances post-punk et dream pop. Some of them are atrocious!!! Pun definitely intended. Cocteau Twins were a Scottish gothic rock and dream pop band whose discography contains nine studio albums, seven compilations, and numerous EPs and singles. Cocteau Twins produced an extensive body of work between 1982 and 1996—over 140 songs—and appeared in or were the subject of a handful of collections, collaborations and special appearances. Liz Fraser gets a lot of credit, but what really makes the Cocteau Twins sound is that dreamy guitar of Robin Guthrie. Complete your Cocteau Twins collection. They become a drug that you cannot get rid of, if they are one of the bands favored by Robert Smith, who understands music, and not a little, it makes me more and more sure that I am a marvel. This is in my top 10 albums of all time, and my favorite from the Cocteau Twins. Why ruin it? Cocteau Twins multi-instrumentalist Simon Raymonde stepped to the forefront with his first solo, Blame Someone Else (Bella Union, 1997). Achetez les Vinyles, CDs de Cocteau Twins, et plus encore sur la Marketplace Discogs. Discography. Produced by Ivo Watts-Russell. Scottish post-punk/ethereal/dream pop band formed in 1981 in Grangemouth, Falkirk and disbanded in 1997. The group was founded in the Scottish town of Grangemouth in 1981, when Robin Guthrie (guitar, production) and Will Heggie (bass) met Elizabeth Fraser in the town's disco. Digitally remastered and re-released in 2005 as part of Lullabies to Violaine. Sleeve design by 23 Envelope. Cocteau Twins [ˈkɒktoʊ ˈtwɪnz]1 est un groupe de heavenly voices britannique, originaire de Grangemouth, en Écosse. Recorded at Palladium Studios, Edinburgh, engineered by Jon Turner. "Pearly-Dewdrops' Drops" has the same charting position as The Spangle Maker EP. Liz makes you dream, and music seems to get married with the best female voice. Lullabies to Violaine: Singles and Extended Plays 1982-1996, "Cocteau Twins - Full Official Chart History", "Cocteau Twins announce new 4-CD set, Treasure Hiding – The Fontana Years", https://en.wikipedia.org/w/index.php?title=Cocteau_Twins_discography&oldid=990003304, Creative Commons Attribution-ShareAlike License, compilation album, joint release by 4AD (UK) and, compilation album of 8 BBC sessions recorded between 1982 and 1996, limited edition (10,000 units worldwide), four-disc box set of single and EP tracks, first two of the four discs in box set shown above, last two of the four discs in box set shown above, This page was last edited on 22 November 2020, at 06:42. First band I ever went to see in early 80's... and the first time I left N. Ireland. I'm a fan of Cure, I think listening to both of them, in a playlist where there are only Cure and CT is a unique, magical experience. I have been a fan of the Cocteaus since first hearing their ethereal otherworldly music in the early 80's and their music has lived with me ever since.

domain: artistic


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World Animal Day Grant Update
Last year, we awarded a small World Animal Day grant to the Swaziland Animal Welfare Society.
Below we include a short report written by Margaret Dean-Smith, Chief Executive
'As a result of the Grant made to us by Naturewatch Foundation we were able to print 6,000 Pet Care Brochures. These brochures enable us to disseminate information in both the official languages of Swaziland; English and siSwati.
These colourful brochures cover many aspects of Pet Care and we have already distributed them to local Pet Shops, Private Vets and Government Veterinary Clinics throughout Swaziland.
We will also hand them out whenever we visit schools, hold Fund-raising events or run Workshops, thus spreading the ‘word’ about good Pet Care practices to as many people and areas as possible.
We really appreciate this Grant as it enables us to educate as many people as possible.

domain: pet


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Moonraker Design is a boutique design house focusing on bespoke diamond engagement rings and custom designed jewellery. We usually have a 4-6 week waiting period on custom orders. The process of choosing the perfect diamond or gemstone and finalizing your unique design takes time. Having that in mind you can be assured that every aspect of your request has our full attention to detail.
Visit our online "GALLERY" at the bottom of the page.
Copyright © 2017 Moonraker Design

domain: design


Carl Orff Quotes
3 Sourced Quotes
Elemental Music is never just music. It's bound up with movement, dance and speech, and so it is a form of music in which one must participate, in which one is involved not as a listener bust as a co-performer. It is pre-rational, has no over-all form, no architectonics, involves no set sequences, ostinati or minor rondo-forms. Elemental Music is earthy, natural, physical, capable of being learnt and experienced by anybody, child's play.... Elemental Music, word and movement, play, every-thing that awakens and develops the powers of the soul builds up the humus of the soul, the humus without which we face spiritual soil-erosion.... we face spiritual soil-erosion when man estranges himself from the elemental and loses his balance.
I am often asked why I nearly always select old material, fairy tales and legends for my stage works. I do not look upon them as old, but rather as valid material. The time element disappears, and only the spiritual power remains. My entire interest is in the expression of spiritual realities. I write for the theater in order to convey a spiritual attitude.


Iran's Revolutionary Guards have released 10 US sailors held for entering its territorial waters in the Gulf, state television reported. They were detained on Tuesday after one of their two vessels broke down during a training mission, the US says.
A statement read on state media said the group was released into international waters after apologizing.
It comes at a sensitive time, as the US and Iran try to implement the deal on Iran's nuclear activities. A US official confirmed the sailors' release to the Reuters news agency.
Current and past news stories.


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I earned my degree in business, then worked in a State Capitol, Microbiology lab, and finally, in advertising, where I fell in love with the creative side. I didn't know how to do anything of course, so for the past few years, I've been self-teaching as much as possible; photography, videography, graphics, web design, you name it.
Videography seems to have a gravitational pull on me. I find it easiest to communicate my idea through video. I'm a dslr shooter - currently using the Canon 7D with and soon moving to the 5D mk iii.
- Flickr - Pics! Some business. Some pleasure. All fabulous.