velocityconf: RT @oreillyanimals Vote Instant Wild's Digital Eyes & Ears for Wildlife Protection to win Google Global Impact Award http://t.co/Z0EetiQshZ

velocityconf: Who's up for a 5 minute lightning talk. Last day to get your #Ignite proposal in for #velocityconf CA. http://t.co/pzL0WJtQH5

Netcare St Augustine's and Netcare Kingsway hospitals have been given the green light to reopen.

South Africa's net death rate has decreased amid the Covid-19 pandemic over the past five weeks, despite the 186 deaths thus far, Health Minister Zweli Mkhize says.

Are you dreaming of being your own boss someday, or would you like to bring in extra income? Starting an online business is one of the easiest ways to accomplish this. The biggest challenge of starting an online business can be developing your business plan. If you are struggling to come up with that brilliant […]

Cycling in Tuscany, Italy

Life as an Exchange Student in Northern Italy

Italy - The Cinque Terra, Lake Como and Milan

The Amateur Traveler talks to Ira Bernstein about two lesser visited cities in Italy - Turin and Bologna. Turin has the reputation as the Detroit of Italy because of its long association with Fiat but that nickname does not express the beauty of the city, the warmth of its people nor the quality of its wine. Bologna has a reputation for good food in a country known for its good food which may be one of the reasons that Bologna is known as "the fat". The city is the home of bolognese sauce and lasagna. It also lays claim to one of the oldest if not the oldest university in Europe. Ira takes us on a tour of surprising museums (like the largest Egyptian museum outside Cairo), pivotal history and of course wonderful food. 

Amateur Traveler talks to Matt Kepkes of NomadicMatt.com about his trip to the ancient capitals of Thailand: Ayutthaya, Lopburi and Sukhothai. Before the capital moved to Bangkok it was in each of these three cities as invaders came in from what is now Cambodia and Burma and then were subsequently repelled. Instead of taking the night train to Chang Mai as many tourists do, Matt and his traveling companions explored these cities on the way. In their travels they found ancient temples (some in ruins and some well preserved), wonderful night markets, troops of monkeys, friendly people and a slice of rural Thailand.

The Amateur Traveler talks to Italophile Ira Bernstein who returns to the show to talk about day trips in Tuscany that you can take from Florence. We talk about visiting the walled city of Siena and its famous bareback horse race the palio, viewing the many towers of San Gimignano, climbing the leaning tower of Pisa (after you take the obligatory picture of someone holding it up), and walking on the walls of Lucca. Each town has its own character and attractions. Arezzo has a jewelry museum and the home of the “Father of Humanism” Petrarch. Fiesole is a rich enclave, a suburb of Florence with views of the city and a roman theatre. Montepulciano and Montalcino are both known for their wines. Ira talks about some of the events that the locals enjoy like the palios, flea markets and flag throwing competitions. So even if you just get as far out of Florence as Piazzale Michelangelo there are many places to explore in Tuscany.

The Amateur Traveler talks to Jessica Spiegel of BootsNAll’s Italy blog “Why Go Italy” and the Eye on Italy podcast about one of her favorite cities in Italy: Milan. Jessica tells us about the fashion industry, the spectacular cathedral, and the first mall as well as some quiet neighborhoods and parks. She also tells us how far in advance you should get tickets to see Leonardo di Vinci’s Last Supper, what to expect when you watch soccer in Italy, and what to do when you see movie stars walking down the streets of Milan. We talk about wonderful Italian people and the joys of learning Italian.

The Amateur Traveler talks to Carrie from cruisebuzz.net about a popular vacation spot just off the coast of California, Catalina Island. 

The Amateur Traveler talks to Rebecca about her adopted home of Umbria in Italy. Umbria is known as the green heart of Italy. Perhaps less well known than its neighbor of Tuscany to the North Umbria is a verdant region of hill towns, farms and national parks located in the center of Italy.

The Amateur Traveler talks to Antonello Losito from Southern Visions Travel about his native region of Puglia (Apulia) in Italy. Puglia is the boot of Italy and does not get the same number of tourists as Northern Italy. But Antonello recommends his rugged coastline and mountain towns as a way to see the Italy that “most American’s picture”.

Hear about travel to the South Tyrol region of Italy as the Amateur Traveler talks to Alison and Andrew Cornford-Matheson from CheeseWeb.eu about their recent trip to this lovely region in Northern Italy. This predominantly German speaking region of Italy includes portions of the Dolomites mountain range, a wine road and some great vistas.

Hear about travel to Lazio, Italy as the Amateur Traveler talks to Nancy Parode from about.com and sixtyandme.com about one of her favorite regions in Italy. Lazio is the region of Italy that includes Rome but this episode will feature all the things outside of Rome.

Hear about travel to Naples and the Amalfi Coast in Italy as the Amateur Traveler talks to Beth Rubin from SelectItaly.com about how to plan a trip to this beautiful region. 

Hear about travel to Sardinia as the Amateur Traveler talks to Max Hartshorne from gonomad.com about this off the beaten path part of Italy. 

Hear about travel to Mantua, Verona, Padua Italy as the Amateur Traveler talks to Gina Mussio from ginamussio.com about these wonderful cities in her adopted country.

Hear about travel to the Crystal Coast of North Carolina as the Amateur Traveler talks to Kit Parks from the Active Travel Adventures Podcast about the area.

Hear about travel to the Island of Sicily in Italy as the Amateur Traveler talks to Barbara Riedel from barbaralicious.com about the home of her mother's family and the "home of her heart".

Hear about travel to Mantua, Verona, Padua Italy as the Amateur Traveler talks to Gina Mussio from ginamussio.com about these wonderful cities in her adopted country.

Hear about travel to Barolo and the Piedmont region of Italy as the Amateur Traveler talks to Betsy and Greg Ball from eurotravelcoach.com about this wine region that they love.

Hear about travel to the Le Marche Region of Italy as the Amateur Traveler talks to Chantelle and Giovanni from theitalianontour.com about this off the beaten path destination.

Do you have the equivalent of a will for all your digital possessions—photos, email, online accounts, and all the rest? If not, you should, and we have a book that shows you what to do. Plus, Interesting Thing of the Day readers can save 30% on it.

Digital cameras make it easy to take way too many pictures. Need help sorting, organizing, storing, and managing them? We've got a great book for you—and Interesting Thing of the Day readers can save 30% on it.

At a cost of $7.5 million a patient, they were 16 very expensive field hospitals. Yet, according to NPR, those hospitals are now “stand[ing] down.” You probably remember them from headlines early in the pandemic: makeshift medical centers being assembled at breakneck speed by companies contracted by the Army Corps of Engineers in anticipation of…

The post After Treating Barely Any Patients for a Massive $7.5 Million Each, 16 Emergency COVID Hospitals Are Standing Down appeared first on The Western Journal.

As confetti floated through the air, a baby boy and his family celebrated his final chemotherapy treatment in time to go home before his first birthday. According to KSAZ, young Aaron has been battling a rare form of cancer called acute megakaryoblastic leukemia since he was four months old. Aaron was a patient at Duke…

The post 10-Month-Old Gets Heartwarming Celebration from Hospital Staff After Finishing Chemotherapy appeared first on The Western Journal.

Game designer and publisher Dave Gilbert founded Wadjet Eye Games in 2006.  This interview features conversation about point and click adventure games; digital game development, marketing and publishing; and the relationship between art, passion and real world commerce.

Jeffery Klaehn: How did you first become interested in point and click adventure games?

Dave Gilbert: I played King’s Quest at a very impressionable age! I typed the word “jump” and I saw Graham actually jump, and I was so blown away that I’ve been playing them ever since.

JK: You founded Wadjet Eye Games in 2006 to sell your game, The Shivah, commercially, then moved to pursue game design on a full-time basis and released The Blackwell Legacy, the first in what would become a series of five games.  What are your thoughts on these games and on the market then compared to now?

Dave Gilbert: I am Blackwell Legacy’s biggest critic. It was the first game I wrote with the intention of selling commercially – The Shivah was originally freeware, so I don’t count it – and it shows every inch of my inexperience. The gameplay is clunky, the story is told in three giant infodumps, and the main characters weren’t very likable. But that said, I know with absolute certainty that it was the very best game I could have made with the experience, resources and time I had available. So I stand by it.

As for the market, everything is different. Back in 2006, indie games in general were a very new thing. Read the rest

Including a shoulder-brush with a livid Steve Waugh, Rob Houwing remembers witnessing SA's heroic finish to an otherwise stormy 2001/02 Australian tour.

Ian McKee, founder and CEO of Singapore-based Vuulr, explains the value of blockchain technology—and the threat it poses to the structure of traditional digital advertising.

Patrick Jones, global vice president and general manager of partnerships for Oracle Data Cloud, discusses how he expects agencies and brands to evolve their use of audience data sets this year.

Omer Golan, founder and CEO of Outernets, discusses how artificial intelligence can improve the digital out-of-home industry.

Video: Merlin Mann - "Time & Attention Talk (improvised)"

Audio (mp3): "Merlin Mann - 'Rutgers Time & Attention Talk'"

This is a talk I did at Rutgers earlier this month. I kinda like it, but for a weird reason. Something something, perfect storm of technology Ragnarok, and yadda yadda, I had to start the talk 20 minutes late with no slides. Nothing.

So, I riffed.

And, I ended up talking about a lot of the new stuff you can expect to see in the Inbox Zero book—work culture, managing expectations, the 3 deadly qualities of email, and one surprising reason email's not as much fun as Project Runway.

Some people liked it. I think. I liked it. I hope you do, too.

Here's the slides I would have shown. ;-)

Many thanks, again, to my great pal, Dr. Donald Schaffner, for bringing me in for this visit. I had a great time and met some fantastic, passionate people. Much appreciated.

update 2010-04-27_13-50-00

Apologies—my friends at Rutgers (inexplicably) have placed this video under lock and key. Fortunately, I have a lock-picker called Firefox. Samizdat video available soon...

update 2010-04-27_14-42-24

Yay, fixed! Many thanks to my hero, Jesse Schibilia.

Research Event

Event participants

Glada Lahn, Senior Research Fellow, Energy, Environment and Resources Programme, Chatham House
Greg Shapland, Associate Fellow, Middle East and North Africa Programme, Chatham House 
Moderator: Sanam Vakil, Deputy Director and Senior Research Fellow, Middle East and North Africa Programme, Chatham House

Research Event

Event participants

Chatham House and Routledge, Taylor & Francis are launching the Journal of Cyber Policy on 2 July.

Dr Paola Subacchi, director of Chatham House’s International Economics Department, has been awarded the prestigious Order of the Star of Italy.

Members Event

Event participants

Undersecretary Myrna C Cabotaje, Public Health Services Team, Department of Health, Philippines

Alan Jope, CEO, Unilever

Josephine Karwah, Mental Health Advocate

Dr Dévora Kestel, Director, Mental Health and Substance Use Department, World Health Organization

Chair: Robert Yates, Director, Global Health Programme and Executive Director, Centre for Universal Health, Chatham House

Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1Cre-mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1Cre;Meis2f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.