text:
A round-up of recent news in clean technology and cleantech investment.
Electronic cooling tech firm 4energy has raised £2m from Carbon Trust Investments and Catapult Venture Managers. The Nottinghamshire-based company produces passive cooling systems that can replace energy-intensive air-conditioning in ICT equipment rooms. Its CoolFlow system promises energy savings of up to 90% over conventional AC.
4energy has a solid list of domestic clients including Vodafone, EDF Energy and Scottish Power Energy Networks, and is eyeing international markets - especially those with a growing telecom infrastructure such as the BRIC economies.
Energy monitor group Green Energy Options (aka GEO) has raised £800,000 growth funding. Angel group Thames Valley Investment Network contributed £250k, with match funding from Bank of Scotland Corporate's growth capital wing, and the remainder from individual investors.
Cambridge-based GEO produces a range of displays and accessories which allow households to monitor and reduce their energy use.
UK rival Onzo raised £2m from Scottish and Southern Energy (SSE) and Sigma Capital Group in April.
French windfarm developer Valorem has closed a Euro16m second round from existing shareholders including Avenir Enterprises Gestion, Crédit Agricole Private Equity and Grand Sud Ouest Capital. The funding will allow the firm to finance its own wind projects - it's aiming at over 100MW installed capacity by 2010.
A clean(ish?) investment from an oil-and-gas specialist - Energy Ventures has pumped NKr45m (Euro5.7m) into tech developer Energreen. The Norwegian firm is developing a turbine, dubbed RotaChoke, which can generate energy from pressure drops in fluid process systems. The tech will allow on- and off-shore operations to reduce their own use of fossil fuels, and thus pump out oil and gas in a cleaner and cheaper fashion. It just about makes sense, I suppose...
Meanwhile in Finland, 'personal solar energy harvesting' developer Suntrica has raised undisclosed early-stage funding from state-backed VC Veraventure and Hong Kong's Yellow Dragon Enterprises. Suntrica is developing advanced PV chargers for phones, laptops and suchlike.
German solar supplier Concentrator Optics has raised an undisclosed round from Capricorn Venture Partners, via its Capricorn Cleantech Fund. Concentrator Optics, as the name suggests, produces optics for concentrating photovoltaic applications, and is building a factory for its Fresnel lenses (with a nominal capacity of 100MW) in Marburg, Germany.
German fuel cell start-up Enymotion meanwhile raised undisclosed seed funding from our old friends at High-Tech Gründerfonds and Zukunftsfonds Heilbronn. The firm is developing fuel cell systems for mobile leisure use which can be powered by conventional fuels such as camping gas.
A couple of vehicular investments from the States. Plug-in hybrid developer Fisker Automotive has completed a $65m third round led by a new Qatari investor, and also taking in current shareholders Palo Alto Investors and KPCB.
And electric bus tech developer Adura Systems received a further cash injection from New Frontier Renewable Energy as it works on raising its first full $10m round.
Solar tech company EnPhase closed a $15m third round led by RockPort. The California firm produces a compact inverter to convert DC from small-scale PV into AC for the grid.
Fund news
London-based cleantech VC WHEB Ventures has announced a first close of its second dedicated fund at £57m. Committed investors include family trusts for the Rothschilds and the Bamfords of JCB fame. WHEB is aiming to raise a total pot of £150m, which should make it Europe's largest dedicated cleantech VC fund.
New Nordic investor Sustainable Technologies Fund has raised Euro58m from institutional investors for its maiden fund. The fund will provide expansion capital for Nordic firms in renewable energy, energy efficiency, renewable materials and green chemicals, recycling and purification technologies.
Fortis Investments is raising a Euro400m clean energy fund. The fund, anchored with Euro50m from its Belgian bank parent, will focus on project finance for wind, small hydroelectric, solar PV and biomass installations.
Further reading
Some slight encouragement in the UK government's new paper on manufacturing strategy. The paper outlines proposals (albeit in fairly wafty terms) to support the move to the low-carbon economy 'as the response to climate change creates both new challenges and opportunities for manufacturing firms':
Recent analysis suggests that there are significant opportunities for UK manufacturers to benefit from developments in clean technologies and products[...]
Ernst and Young identify the ability to attract capital including venture capital due to the existence of strong financial markets; the supply of high quality services to start and promote a new business (including strong software and business/management services); and the presence of a sophisticated and high tech manufacturing base as the key characteristics underpinning current advantage in these sectors.
The paper also proposes measures to remove barriers to wider deployment of renewable energy, and to support electric and plug-in hybrid vehicles.
A new report from Greentech Media and the Prometheus Institute on the growth of thin-film solar - they're predicting eight-fold growth to over 4GW worth of production by 2010, with amorphous silicon making up half of that. Cadmium telluride may end up as a niche tech, while CIGS is yet to reach breakthrough.
And from the Cleantech Forum in Washington DC - cleantech insiders weigh Wall Street turmoil. While the weight of money in funds means that VC is likely to continue to roll, project finance looks to get tougher and the IPO route doesn't look at all cheery. Interesting times, to be sure...
Tuesday, 16 September 2008
Clean Sweep 45
A round-up of recent news in clean technology and cleantech investment.
Posted by Tim Chapman at 16:43


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Ginshar / Ginsharians
The Ginshar system is located in the Adar subsector of the Hinterworlds (0501). It is listed as an amber zone, with good reason. Ginshar is home to a very secretive offshoot of Humanitii.
Ginshar is a totally artificial world. The population is entirely made up of manufactured, synthetic humanoids and robots. The synthetics are manufactured with an artificial genetic material called TXNA or Tri-Xeno Nucleic Acid. It is a triple helix strand of genetic material consisting of the normal, but slightly modified DNA with a third strand of genetic material added. This third strand is made of artificial nucleic acids that act as electronic circuits giving a Ginsharian the genetic equivalent of an incredibly powerful organic computer system within their body.
It also acts, with their carefully designed DNA, to build in all sorts of added "features" and non-human attributes. This means that Ginsharians, by design, can perform all sorts of activities, have senses, or other attributes that exceed human abilities significantly.
The down side to this design is that all TXNA Ginsharians are dependent on taking anagathics continuously. Should a Ginsharian not have access to anagathics they will suffer a complete breakdown of their genetic code and in a short time die a horribly painful death.
Ginsharians could have any of the following enhanced abilities, but not limited to:
Enhanced sensory abilities
Ability to interact directly with electronics and computers.
Ability to gain energy from electrical and electronic systems.
Massive memory capacity.
Ability to act as a very powerful computer.
Enhanced abilities like strength, speed, etc.
Ability to work / survive in environments that humans can't without specialized clothing, respirators, or other equipment.
Have specialized abilities like producing a particular hormone to cause others to react to them, or enhanced healing of wounds, etc.
Ginsharians are "programmable" like a computer, but they can also learn like a human can. Most can switch between a more machine-like mode and a very human one as the situation requires.
Ginshar itself is an amorphous thing. Ginsharians make up something like a hive mind in groups and also are linked to a higher intelligence called "Ginshar."
Each "model" of Ginsharian could be manufactured in any needed quantity. A particular model looks the same as all others of that model (like identical twins). The use of anagathics means that they don't really age over their lifetime.
All Ginsharians have a four letter designation followed by a number showing their sequence of manufacture for their model. Examples: Adam 33 or Lisa 198.
Written Ginsharian is an octal numeric language consisting of blocks of 8 numbers (0 to 7). These translate into words and numbers for Ginsharians who can decode such a language using their built-in computer system. It also makes Ginsharian almost indecipherable to anyone else.
Ginshar manufactures non-TXNA synthetic humans and other species for off world buyers. These are expensive but exquisitely well made. Manufacture of such things as slaves, "sex toys," and other sorts of almost universally illegal types are available from them. Nobles and the filthy rich who have "odd proclivities" are noted customers of this black market merchandise.
Another specialty is mercenary armies. An advantage of a Ginsharian mercenary unit is they are completely loyal, incorruptible, and extremely efficient as soldiers. They are known to be utterly fearless and capable of fighting far in excess of human troops.
They are also anything but cheap. As bodyguards they are the best money can buy.
Ginshar also manufactures a range of products like exotic food, pets, plants, etc., that are highly desired because of both their rarity and physical qualities. Ginsharian liquors and foods are something any noble or well-heeled individual would want to have to impress their peers.
Another specialty is high tech drugs. Ginsharian anagathics are some of the best available anywhere. They also make numerous illegal drugs for buyers with money.
Ginsharians are extremely secretive. It is rare for someone to run into more than one of a particular model anywhere. Outsiders generally only meet with a handful of specialized models designed specifically for interacting with what Ginsharians universally call "Aliens." That for them is as much a pejorative as description.
Ginsharians often display limited ability to interact with outsiders (Aliens). They tend to be reclusive and introverted. They also tend by Human standards to be exceptionally, even stunningly, good looking.
They also tend to only want to perform the tasks and activities for which they were designed. They are reluctant to learn new things but will as the situation dictates.
Ginsharians prefer a very clean, sterile, artificial environment. Nature, outdoors, or a dirty, gross environment repulses them. As a rule they are agoraphobic and "neat freaks." With food it is no different. They prefer artificial things like flavored protein cubes.
The Ginshar system is an Amber Zone because there is a massive, instant, and highly negative response to any ship arriving in the system uninvited. Those who are visiting Ginshar for some reason (usually business), will find they are escorted everywhere they go. They will have limited or little interaction outside a few specialist models that are designed to deal with them. Everything is secret and if Ginshar suspects a visitor is spying, even if something innocuous, they will be killed and all traces of their existence erased.
The system is extremely well defended and anything short of a major invasion would probably fail. Worse, Ginshar's capabilities with computers and electronics along with their spy network would almost certainly result in a massive attack on their antagonist's systems with the resulting heavy damage or destruction of them.
Ginshar is well known for its ability to hack and attack computer and electronic information systems with larger intelligence agencies. Spying is one of their fortes.
On the plus side for "Aliens" Ginshar shows little or no desire to be aggressive, expansionist, or oppressive to others. It takes a direct threat to them to get them to respond and then the response is total.
Ginshar is well known within about 10 to 20 parsecs as a place you don't mess with and Ginsharians are taken the same way generally. They are as much things of lore and legend at truth. As a rule the higher one goes in the intelligence community or society the more likely one is to have some actual knowledge of Ginshar. For the average person it is one of not knowing or of being in fear of them.
Ginshar is extremely inquisitive. Ginshar wants to know everything. To that end, Ginshar is insidious at espionage. Ginshar manufactures lots of synthetic humans (and other species) with normal DNA (more or less) that are created specifically to be inserted into other societies for the purpose of collecting information and sending it back to Ginshar.
The use of micro- and nano- machines to move this information is common. These hitch a ride on ships and cargo moving towards Ginshar to be collected periodically. Nowhere within about 100 light years (30 to 40 hexes) of Ginshar is safe from this sort of thing. It may extend beyond that.
Ginshar has outposts on several nearby systems that act as outlets for their activities and products such as a sky city on Boilingbrook or a military outpost on Kasasha.
Ginsharian ships and military action:
Normally these are matt black, unmarked, and will not respond to other vessels. If they do it is usually a direct threat to leave them alone or else. Ginsharian mercenaries rarely give warnings to anyone they perceive as a threat or enemy. An attack on one Ginsharian is an attack on all Ginsharians and one should expect any Ginsharian to be armed at any and all time.
That means if you attack a Ginsharian ship and they are able to get a message / information about you back to Ginshar expect to be hunted down and annihilated by their forces eventually. They are relentless and never forgive their enemies.
Game notes:
Where Ginsharians are interesting in game terms include:
The players discover one of their "experiments" or spies on a world and figure out what they're doing. Mess with the experiment / spy is messing with Ginshar.
The party has to interact with Ginsharian mercenaries or others for some reason.
The group comes on a lone Ginsharian stranded needing help. The Ginsharian in question has abilities that cause the group serious problems unintentionally.
A high ranked noble / person has a Ginsharian "product" with him / her and wants discrete, private, passage, etc. The product is highly illegal and the crew expendable beyond transporting this person somewhere. The "product" could cause additional problems…
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Manual Dispenser is an attractive, high capacity and versatile wall mounted dispenser.
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Cost Effective, Easy to Use, Prevents Waste.
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text:
Sunset on the beach at Crystal Cove, Ca
Every year, around the time of our anniversary, Clay and I gather all our children to celebrate family day. We feast together, share in our love and then spend hours of serious commemoration of all the ways we have seen the fingerprints and faithfulness of God in our lives that year. And so, at the last moment, we were able to get all of us reasonable tickets to California where the boys are, to celebrate our history, one more year. It is a day of great import to each of us and our souls, sharing together in God's faithfulness each year in our lives--a day to leave memorial stones for others to see, and to worship Him.
A two bedroom apartment at the beach was our house of worship. Off to bed after our flights, before our day of celebration would dawn.
At 5 a.m, Friday morning, Sarah crept quietly into our darkened bedroom and said, "There was a call on your cell phone in the living room a few minutes ago."
My brother had called, "Mom just passed away and I wanted you to know."
Waiting each day, wondering when it would be, I was anticipating my mama passing from this life into the presence of Jesus.
Now, it was final, there would be no more opportunities for words of life between us, no sweet caresses against squishy warm cheeks, no putting to rest any untimely issues, no more moments to bless or take away curses. Her opportunity to live a story was now over. This thought entered my mind. Others began to push their way in---
A week before, my family and friends had cleaned my home, placed china on our tables, placed flowers into vases and cooked our homemade food--we had prepare for friends who were coming to our home to be blessed.
I was wondering, if God who helped me to be able to conceive of "preparation for loved ones", the one who said he "was going to prepare a place for me in heaven to receive me", had prepared a welcome committee that morning for my mom. Perhaps my dad would be there, my oldest brother, her mom and dad and maybe even her grandparents. I liked thinking of Jesus preparing for a welcome for my mama. It made me smile to think about her feeling well again, smiling, hugging, seeing those who had gone before her who had shared in her life and loved her.
Memories began to flood my heart, the birthdays were always documented--the trail of pennies from my bedroom to a pile of presents when I awakened, that stands out from all the rest. Lighting candles each evening and putting on music and painting on bright red lipstick to prepare for the welcome, as my dad came home from a hard day at work. Christmases, Thanksgiving feasts, delectable homemade food in general and the multitudes of times we had girl dates together, the ways she served me as an asthmatic child, singing songs to me in the dark nights of difficult breathing, comforting, the way she gently played finger games with me in church to pass away the long minutes of the sermons, Her willingness to listen to me many hours throughout the passages of childhood, the little welcome signs that greeted me every time I came home from college or for holidays.The moments she invested, the thoughtful ways she mothered, for these I will always be deeply grateful.
Nathan and me, cherishing moments together on the beach at sunset.
A new thought came to me, "How very wonderful for me to get the news here in California, at the beach, with my sweet family, my four kids and Clay, where those I loved the very most could be together. And a place where I was not accosted by the demands of normal life, phones, dishes, duties, but where I could be away to put to rest this history of me and my mama. How considerate my sweet Father was to my very demanding life.
What surprised me were the demons that crept into my brain and heart. They raised their ugly heads and voices to remind me of issues that could never be addressed again or put to rest. I felt some deeply painful fingerprints on my heart, whose imprints will be there forever. I am an adult on the outside, but inside, my heart is that of a child, the heart that bears all the goods and bads, the memories, the impressions, and it was there that daunting pain raised it's head and whispered of the wounds inside where no one could see.
And so a part of the weekend, in the midst of family day with all 4 children and a precious new friendship for our family, and the sharing of life and meals and the ponderings of God's faithfulness to us,and planning the timing for the memorial services, I spent hours on the beach, pondering, reviewing what was there, observing what remained, in the hours between 6 a.m. and 8 each morning miles and miles on the almost abandoned beach, I walked through the hallway of the remains of my pain, and found His presence speaking to me there.
And the grace that we share in Jesus is that His redeeming love renders the the voices of darkness silent and brings the sure comfort of His healing touch.
These words He left me, "Honor your mother." "Love covers over a multitude of sin. Love is the perfect bond of unity." "Love one another." "The steadfast love of the Lord never ceases. His mercies never come to an end. They are new every morning. Great is thy faithfulness.
But, sometimes we need time to hear His voice, to gain His perspective and to see His way. Healing is not always instantaneous and so He had already planned that I would need this, and prearranged for me to be on a beautiful beach, away, to put to rest the legacy of this heritage--while helping me to clearly understand, that now, I must be more intentional than ever about the legacy I will one day leave--for all women, will leave a legacy--one that is intentional or one that is passive, but nonetheless remains.
And in this moment, I realized how definitively God had created this story to give me a passion to help other mothers understand the eternal impact of their lives. This relationship of mother to child matters deeply, personally, and will potentially have an impact forever.
And so I pondered, how do I want to leave a legacy? What legacy do I want to leave? What legacy will I leave?
My arena to bring life--Nathan, Clay, Sarah, me, Joy and Joel--Family Day 2011
And so, I find that because of my own life's history and the ways He has placed His call on my life, He has given me such a joyful heart-filled opportunity. I know more deeply than ever, that a mother only has one life to tell a story to her children of His grace. I understand more clearly, only these moments today, a gift, to face life head on with faith, finding strength from Him to be a champion for His kingdom. Today, I have the seconds left to bless my children, believe in their dreams, give love to their hearts, speak words that will remain forever. I want to live fully engaged in all that He has planned for me--to give as generously with all of His treasures today, because one day I will have no more time to sing, to love, to worship and to bring life. And so in the living of this day, and seeing my moments as gifts of opportunity to bring life and light, I will choose to face this day with courage, strength, hope, grace, love and a deeply grateful heart.
This day with my baby girl attending her first college class, when Clay will meet with a surgeon to find out when he will need to have his surgery and what it will mean, Nathan needs my help in his projects asap, all the bags need to be unpacked, travel to Texas arranged, but in this day, I will rejoice for the life that is left to me to invest today, for eternity and for His kingdom.
But always, I will carry my mama with me in my heart, and remember her last words, spoken a couple of months ago, "I love you. I am proud of you." A legacy of life inserted in the midst of her dying--that I can dwell on when I need a place of her touch to revisit.


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text:
The History of Mitsubishi Forklifts
Since 1870, Mitsubishi has developed a fleet of well-engineered trucks, all of which maintain an excellent reputation for dependability and productivity. Mitsubishi is proud of their ability to keep all of their equipment in peak condition by an international network of fully trained dealers and service technicians. Mitsubishi began in Japan, but has since expanded and grown to global markets. In fact, Mitsubishi has evolved into aeronautic, manufacturing and electronics.
Mitsubishi boasts a North American headquarters in Houston, Texas utilizing over 600,000 square feet of commercial space, advanced robotics and superior supply chain practices to assure timely and cost-effective delivery of all products and goods. Investing in a used Mitsubishi forklift or used Mitsubishi lift truck could be wise, as they have been proven to be resourceful in a variety of endeavors. Here are some of Mitsubishi's most noteworthy and popular forklifts used commonly by many customers and warehouses today:
Notable Mitsubishi Products:
• FB 16/18/20 Series Electric Counterbalanced Three-Wheel Forklift
• FBC 16/18/20 Series Electric Counterbalanced Cushioned Wheel Forklift
• ESR 15/18/20/23 Series Electric Single Reach Truck
• PW/PWT Series - Power Walkie Truck Device
• FG Series Internal Combustion Cushion Tire Forklift
• FD70 Heavy Duty Internal Combustion Pneumatic Tire Forklift
Why Choose Mitsubishi Forklifts?
Mitsubishi is well known for superior engineering and design. Their specifications are known to deliver superior dependability and fuel efficiency. In fact, Mitsubishi's FD70 Heavy Duty series is a one-of-a-kind model, a forklift equipped to lift up to 15,000 pounds and deliver improved efficiency compared to its previous generation. This is a testament to Mitsubishi's assurance of quality and improvement of their goods and services. Many Mitsubishi products feature a Perkins 840 Supercharged engine, giving the machine the power it needs to perform at its best.
Another great product offered by Mitsubishi Forklifts is the PW series. The PW, or PWT, are power or manual walkies that can lift pallets of up to 6,000 pounds. These walkies have some of the smallest heads in the industry, further improving handling and maneuverability in the tightest and narrowest of turns and corridors. In addition, the PWR power rider has a wide array of features ideal for any novice operator. The most notable of these is the suspended operator platform, utilized for long and heavy loads. These devices offer an advanced corrosion protection package, rendering these products impervious to many of the hostile conditions found in many warehouse and manufacturing settings. This package includes galvanized chassis, pull rods, load wheel linkage and forks.


text:
Enoteca – Delicious recipes
Olive Paste And Blue Cheese Canapi
– 1/4 pound pitted cured black olives
– 1 large clove garlic, mashed to a paste or put through a garlic press
– 2 tablespoons pine nuts
– 3 tablespoons olive oil 1/4-inch slices long crusty loaf bread
– 1/4 pound blue cheese (Cabrales, Roquefort, or Gorgonzola), or more Black olives, for garnish
– Place the olives, garlic, pine nuts, and olive oil in the bowl of a processor and chop as finely as possible.
– Spread thinly on bread slices. Cover with blue cheese.
– Decorate with a piece of black olive.


text:
I have been busy trying to grow my custom portrait business Fantasy Photos & my freelance graphic design career as Paul Micarelli Design. I am also working for my fiancée's family, doing the marketing & promotion for their home heating oil company- 5 Star Heating Oil. Despite the feeling that I don't have nearly enough time in the day, I also want to keep my general creative spirit flowing.
By sharing my artwork, I hope to maintain this aspect of my creativity. I have a huge archive of my images that I can post & offer for sale.
This week I dropped off some prints of my abstract art to a local gallery.
One of the images was this one, that I had not uploaded to my online album yet:
by Paul Micarelli
"Metal Melt"
by Paul Micarelli
***Buy Prints & Products With This Design***:
The Monkey Buddha Bazaar:


text:
Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress.
Mitochondria are found in almost all eukaryotic cells and play a role in processes such as ATP production, Ca2+ homeostasis, lipid synthesis and apoptosis signaling (Nunnari and Suomalainen, 2012). Mitochondria are organized as a network that undergoes continuous events of fission and fusion, processes that are crucial for their cellular function (Chen and Chan, 2009). The balance between the fission–fusion cycles is essential, as its disruption is implicated in human neurodegenerative diseases (Lin and Beal, 2006; Chen and Chan, 2009; Federico et al., 2012). The mitochondrial network responds to changes in physiological conditions, such as starvation and oxidative stress, as reflected in morphological rearrangement that results in hyperfusion and fragmentation, respectively (Fan et al., 2010; Rambold et al., 2011). Additionally, the mitochondrial network responds to various cues by changing its intracellular positioning, supplying local ATP demand and providing a localized signaling platform (Tait and Green, 2012; Desai et al., 2013; Sheng, 2014).
Mitochondrial motility is primarily based on microtubules (MTs), utilizing plus-end-directed kinesin motors and the minus-end-directed dynein (Pilling et al., 2006). However, the actin cytoskeleton has also been shown to be involved in mitochondrial motility, localization and cellular distribution (Bradley and Satir, 1979; Chada and Hollenbeck, 2004). Myo19, an actin-based molecular motor, was discovered as a new mitochondria-localized myosin in vertebrates. Myo19 localizes to mitochondria through its C-terminal tail domain and nearly doubles the mitochondria motility when ectopically expressed (Quintero et al., 2009). The finding of a myosin that localizes to the mitochondria suggests that it acts as the link between mitochondria and the actin cytoskeleton. Knockdown of Myo19 by RNA interference results in apparent defects of mitochondrial segregation during division and incorrect movement of the mitochondria to the spindle poles during anaphase (Rohn et al., 2014).
The interaction between the actin cytoskeleton, myosin motors and their function in mitochondria dynamics, morphology and cellular localization is now beginning to emerge (Korobova et al., 2014). A new function for actin-based motors could emerge as regulators of cellular adaptations to stress, linking actin cytoskeleton remodeling and mitochondria (Jayashankar and Rafelski, 2014). These recent studies demonstrate an essential role for both actin cytoskeleton and myosins for mitochondria function in homeostasis and pathological conditions.
To this end, we examined Myo19 involvement in the mitochondrial network response to stress conditions induced in human U2OS cells. We discovered that Myo19 localizes with mitochondria to tips of starvation-induced filopodia in a process that depends on its ATPase activity and the actin cytoskeleton. We show that RNA interference (RNAi)-mediated knockdown of Myo19 attenuates the formation of starvation-induced filopodia. Deletion of 45 amino acids in Myo19 tail domain (Myo19Δ851-895–eGFP) impairs its localization to the mitochondria. We further dissected Myo19 binding to the mitochondria, revealing that it is a monotopic membrane protein anchored to the OMM with both the N- and C-terminus facing the cytoplasm. To our knowledge, this is the first vertebrate myosin that is directly bound to a membrane.
Glucose starvation induced localization of mitochondria and Myo19 to foci at cell periphery protrusions
Mitochondria function as an intracellular biosensor that responds to environmental changes, stress cues and physiological stimuli (Jayashankar and Rafelski, 2014). Hence, it is very intriguing to test the Myo19 interaction with the mitochondrial network dynamics and morphology in cells under stresses, such as starvation, that induce mitochondrial responses. We therefore ectopically expressed Myo19 fused to eGFP (Myo19–eGFP) in U2OS cells, and followed Myo19 and mitochondria localization in response to glucose starvation. Under complete medium conditions, Myo19 localized to mitochondria and there was also some diffuse cytosolic appearance, which might be due to the ectopic expression, similar to in previous work (Fig. 1A). Notably, expression of Myo19–eGFP caused mitochondria to clump together into globular structures in a motor-dependent manner (Fig. S1A, white arrow; Fig. S1B). In contrast to complete medium conditions, glucose starvation of U2OS cells resulted in the localization of Myo19 together with mitochondria to foci in protrusions extending from the cells (Fig. 1A). We quantified that >80% of Myo19–eGFP foci were positive for mitochondria by calculating the ratio between the number of MitoTracker-Red-stained Myo19–eGFP foci and the number of Myo19–eGFP foci (Fig. 1A, n=160 from 14 cells). Notably, these protrusions formed randomly around the cell periphery, showing no preference of directionality, which is most likely due to missing cues such as mechanical forces or chemotactic molecules (Petrie et al., 2009). The localization of Myo19 to the starvation-induced foci depended on an active full-length Myo19 (Fig. 1A). Neither eGFP alone, Myo19 tail (Myo19824–970–eGFP) nor ATPase-dead full-length Myo19 (Myo19G135R–eGFP) (Adikes et al., 2013) were able to localize to the protrusions, linking Myo19 enzymatic function to protrusion localization. To test whether the localization required a mitochondria-binding motif, we repeated the experiment with a cytosolic mutant of Myo19 (Myo19Δ851–895–eGFP), which was able to localize to these protrusions; however, no mitochondria colocalized with this deletion mutant, demonstrating the requirement for mitochondria-binding motif for colocalization with the mitochondria (Fig. 1A). Verification of expression of the Myo19 constructs was performed by western blotting (Fig. S1C). Supplementation of the starvation medium with glucose completely prevented both protrusions and foci formation, indicating that it is a response to glucose starvation rather than other nutrients (Venter et al., 2014) (Fig. 1C).
We further examined the growth dynamics of these starvation-induced protrusions and the emergence of Myo19 foci within them by performing live imaging of starved cells ectopically expressing Myo19–eGFP (Fig. 2). Most of the foci formed within the cell boundary at time zero of imaging (+20 min of starvation), which is marked by yellow line (70%, n=100 from 14 cells), whereas fewer extended beyond the cell boundary (30%, n=100 from 14 cells). However, we cannot conclude from these experiments the order of events between protrusion formation and Myo19 foci dynamics. The relationship between the protrusions and Myo19 foci can also be seen in time-lapse DIC images, which clearly show the linkage between them (Fig. S2A). Similar protrusions were observed in wild-type (WT) cells under starvation (Fig. S2B; see also Fig. 5F).
To this end, we demonstrate quantitatively the link between Myo19, its mitochondria-binding motif and glucose starvation to localization to starvation-induced foci formation in the cell periphery protrusions. Moreover, these observations raised the notion that Myo19 might influence these protrusions and prompted us to test the involvement of the actin cytoskeleton in these structures.
The actin cytoskeleton is essential for starvation-induced Myo19 foci formation
To support our results that an active motor is required for Myo19 foci formation, we tested whether disruption of the actin cytoskeleton would prevent their formation. Treating Myo19–eGFP-expressing cells with 0.2 µM latrunculin B (LatB) 30 min prior to starvation prevented the foci formation (∼70% of the cells showed less than two foci), further supporting that Myo19 foci formation is through the actin cytoskeleton (Fig. 3A). Alternatively, we show that nocodazole treatment of Myo19–eGFP-expressing cells induced Myo19 foci formation under complete medium conditions (>80% of the cells). This is similar to what is seen in cells ectopically expressing mDia1 and treated with nocodazole, which show a strong shift towards actin-based mitochondrial motility (Fig. 3B) (Minin et al., 2006). However, the molecular basis for this is still unknown.
Starvation-induced protrusions possess filopodia markers
We had strong evidence that the foci are related to the actin cytoskeleton. Therefore, we tested the starvation-induced localization of Halo-tagged Myo19 (Myo19–Halo) with actin–eGFP in live cells or Alexa-Fluor-488–phalloidin-stained actin in fixed cells, revealing that Myo19 foci localized at the tips of actin protrusions (Fig. 4A, inset and line profile; Fig. S3A). To characterize these actin protrusions, we tested Myo19 localization with focal adhesion and filopodia markers, as these structures might provide further insight on the nature of these protrusions. Vinculin and paxillin were used as markers to test whether Myo19 foci are related to focal adhesions. Ectopically expressed Emerald–vinculin localized to the base of the protrusions; however, Myo19 was localized further towards the tip (Fig. S3B, insets and line profile). Notably, expressing paxillin–eGFP inhibited protrusion formation, in accordance with published literature; however, it was not localized with Myo19 in the few protrusions that formed (Fig. S3C, insets and line profile).
The protrusions resembled filopodia in their structure, therefore we tested Myo19 colocalization with the filopodia markers fascin and Drf3 (also known as mDia2), revealing that these actin protrusions are indeed growing filopodia and that Myo19 is present at their tip (Fig. 4B, inset, line profile). Glucose-starvation-mediated induction of filopodia formation is not limited to U2OS cells, as we also show that this also occurs in the HeLa cell line (Fig. S3D), suggesting that this is a universal phenotype. This finding strongly encouraged us to test the contribution of Myo19 to filopodia formation.
Myo19 is a de novo effector of starvation-induced filopodia
To examine the contribution of Myo19 to filopodia formation, we utilized RNA interference to knockdown Myo19 from U2OS cells and follow starvation-induced filopodia formation. We ectopically expressed fascin–eGFP in these cells, allowing us to follow filopodia formation (Fig. 5A; Movie 1). Interestingly, filopodia formed both at the dorsal side of the cell and at the cell periphery. However, Myo19 localized only to the peripheral filopodia, but not to the dorsal filopodia, therefore only these were measured (Fig. 6A). To correctly measure the filopodia length using wide-field fluorescent microscopy, we verified that most of the peripheral filopodia were parallel to the plane by measuring their angle at the z-axis to be <2° (Fig. 6B). Evidentially, knockdown of Myo19 (∼80%, Fig. 5B) resulted in significantly fewer (from 42±7.9 to 24±10.8 filopodia per cell, mean±s.e.m.) and shorter filopodia (from 2.9±1.22 µm to 1.9±0.74 µm in length) (Fig. 5C,D). In addition, we quantified the filopodia length distribution and observed a clear shift towards shorter filopodia in Myo19-knockdown versus mock-treated cells (Fig. 5E). The small interfering RNAs (siRNAs) utilized to knockdown Myo19 were targeted against the 3′ UTR, allowing us to confirm that the effects are specific to Myo19 by performing a rescue experiment, where we knocked down Myo19 and ectopically expressed fascin–eGFP and Myo19–Halo. The rescue reversed the knockdown of Myo19 as measured by restoration of starvation-induced filopodia number to 39±6.5 filopodia per cell and length to 2.8±0.93 µm, similar to in mock-treated cells (Fig. 5D). Moreover, the rescue restored the filopodia length distribution to a similar distribution to that of mock-treated cells (Fig. 5E).
Furthermore, we observed two differences between the mock- and Myo19-siRNA-treated cells. Mitochondria were present in a minority of the filopodia of mock-treated cells, which were completely absent from filopodia of Myo19 knockdown cells (Fig. 5F, arrow). Additionally, Myo19 knockdown cells had visible patches of fascin–eGFP at the cell periphery (Fig. 5G), which might represent a possible failed filopodia formation site. Comparing the rate of filopodia growth between mock-treated and Myo19-knockdown cells indicates that Myo19-knockdown cells feature a pronounced longer lag before reaching a similar steady-state rate of filopodia growth (0.072±0.032 µm/min and 0.079±0.032 µm/min, for Myo19-knockdown and mock-treated cells, respectively), suggesting that Myo19 role is important for the dynamic development of filopodia (Fig. 6C).
We tested the effect of Myo19 knockdown on the mitochondria network morphology; however, we did not notice any major changes compared to mock-treated cells (Fig. 5H). Our collective observations strongly implicate Myo19 in starvation-induced filopodia formation. We next pursued the characterization of the physical linkage between Myo19 and the mitochondria.
Endogenous Myo19 co-purifies with mitochondria
To examine the nature of the physical interaction between Myo19 and the mitochondria, we purified mitochondria from HEK293 cells using differential centrifugation in order to biochemically verify endogenous Myo19 subcellular localization. The differential centrifugation generates three crude fractions: a mitochondria-rich fraction, called the heavy mitochondrial fraction (HMF), a mitochondria-poor, called the light mitochondrial fraction (LMF), and a cytosolic fraction (CYT). Myo19 was present exclusively in the HMF (Fig. 7A). To verify the mitochondrial localization, the HMF was further fractionated in a self-forming Iodixanol density gradient. Mitochondria and ER co-fractionated to some extent (fraction 7–10), as seen by the existence of the mitochondrial and ER markers VDAC and GRP94, respectively. Myo19 only distributed in the fractions of mitochondria and ER, but was absent from the fraction of ER lacking mitochondria, indicating that it interacts with the mitochondria (Fig. 7B). Notably, we did not detect any dissociation of Myo19 throughout the rigorous purification and fractionation procedure, suggesting that this interaction is stable.
Myo19 is anchored to the outer mitochondrial membrane
We next sought to determine the molecular basis underlying the physical interaction of Myo19 with mitochondria. Several possibilities exist for protein–organelle interactions including sub-mitochondrial localization, interaction through a receptor or mediator protein or direct binding to the outer mitochondrial membrane (OMM). The sub-mitochondrial localization of Myo19 was determined by protease protection assay using proteinase K. Myo19 was completely digested in the absence of any detergent, indicating that it resides on the outer mitochondrial membrane (OMM) (Fig. 7C, left). To test the membrane topology of Myo19, we purified mitochondria from Myo19–eGFP-expressing cells, where the eGFP tag is fused to the C-terminus of Myo19, and repeated the protease protection assay. The proteinase-K-treated sample was not recognized by anti-GFP antibody, indicating that the C-terminus faces the cytoplasm (Fig. 7C, right). ENDO G or ATP synthase 5A (ATP5A), both mitochondrial intramembrane space (IMS) proteins, were used as a control to confirm that the IMS was protected from the protease. To address the possibility of protein–protein interactions between Myo19 with an adaptor protein or OMM receptor, we attempted to biochemically extract endogenous Myo19 from purified mitochondria. The Myo19–mitochondria interaction was resilient to high salt (2 M NaCl) and urea (2 M), indicating that the interaction is not limited to electrostatics or to protein–protein interaction but that there is probably an additional binding feature. As a control for the salt and urea extraction, we utilized ATP5A, which can be extracted by salt and urea when mitochondria are subjected to freeze–thaw cycles (Fig. 7D). Interestingly, Myo19 could only be extracted by carbonate extraction from the OMM, suggesting that it is a peripheral membrane protein (Fujiki et al., 1982) (Fig. 7D). Furthermore, Myo19 separated to the detergent phase in a Triton X-114 phase separation of purified mitochondria, whereas the soluble protein GRP94 was found in the aqueous phase (Fig. 7E). These findings suggest that Myo19 is a monotopic membrane protein that does not transverse the OMM, with both the N- and C-terminus facing the cytosol.
Residues 860–890 of Myo19 mediate the interaction with the OMM
To explore the mode of Myo19–OMM interaction, we searched for putative membrane-binding motifs in Myo19 tail domain, which has been shown to localize to mitochondria when ectopically expressed (Quintero et al., 2009). Using the DAS prediction webserver, we identified such a motif between amino acids 860 and 890 (Cserzo et al.) (Fig. S4A). We ectopically expressed Myo19860–890–eGFP as the predicted membrane-binding motif and verified that this motif was sufficient to target eGFP to mitochondria (Fig. 8A). To test whether this domain is essential for Myo19 OMM localization, we ectopically expressed a deletion mutant lacking this region [Myo19824–970(Δ860–890)–eGFP]. This deletion mutant was mostly distributed throughout the cytosol, although it was not fully excluded from mitochondria (compared to eGFP, Fig. S4B). Therefore, we generated a larger deletion, Myo19824–970(Δ851–895)–eGFP, which was further excluded from mitochondria but not completely. These results strongly support the hypothesis that the Myo19860–890 motif is both essential and sufficient for OMM localization; however, we cannot exclude the presence of a minor binding region or other protein–protein region within the tail domain of Myo19 (Fig. 8A).
To test whether the Myo19860–890 membrane motif only dictates OMM targeting or whether it also participates in Myo19 OMM anchorage, we performed biochemical extractions on purified mitochondria from cells overexpressing Myo19860–890–eGFP.
In accordance to our finding in cells (Fig. 8A), Myo19860–890–eGFP was co-purified with mitochondria; however, unlike the endogenous Myo19, it appeared to be more sensitive to extraction by salt. Carbonate completely extracts Myo19860–890–eGFP from purified mitochondria, similar to endogenous Myo19 (Fig. 8B). The difference in salt sensitivity might be due to an insufficient length of the motif meaning it is not fully anchored in the membrane compared to in the WT endogenous protein. Another possibility is that a certain part of Myo19860–890–eGFP is correctly anchored, whereas the rest was present there nonspecifically (e.g. through dimerization of eGFP). Nevertheless, we can conclude from these experiments that this motif is both essential and sufficient for OMM localization.
To further explore the OMM targeting, we examined the motif sequence. Although there is no consensus sequence for targeting proteins to the OMM, it has been shown that a moderately hydrophobic motif flanked by positive residues is required for OMM localization (Rapaport, 2003). Myo19 membrane motif contains a moderately hydrophobic region between residues 864 and 880, flanked by basic positively charged residues (Table S1, flanked by '*'). Therefore, we sought to assess whether Myo19 OMM localization follows these criteria. Point mutations of the basic residues, R882S and K883S, singly or in combination, resulted in a dramatic shift of the intracellular localization of Myo19 to the ER (Fig. 8C; Table S1, Fig. S4C). Increasing the hydrophobicity of the hydrophobic region by the point mutation P865V resulted in dual localization to both the ER and the mitochondria. Analysis of the P865V mutation shows that the hydrophobicity of the motif increased from 1.141 to 1.482 (Kyte and Doolittle, 1982) and, hence, this residue might participate in dictating the correct mitochondrial localization of Myo19 (Fig. 8C; Table S1). Both results are in accordance with similar studies on tail-anchored OMM proteins (Kanaji et al., 2000; Borgese et al., 2001, 2007; Kaufmann et al., 2003; Wattenberg et al., 2007). In addition, the membrane motif with surrounding residues is predicted to contain two α-helices at 856–878 and 882–892 according to the MemBrain webserver (Fig. S4D) (Yang et al., 2013). By plotting this region as a helical wheel using HeliQuest (Gautier et al., 2008), one could observe that the N-terminal portion of the helix has amphiphatic characteristics, which can be found in many membrane-associated proteins (Hristova et al., 1999) (Fig. S4E). Collectively, these results show that the Myo19–OMM interaction is highly specific and mutations in this motif dramatically disrupt this interaction.
Myo19-derived peptide binds to OMM-mimicking vesicles
To characterize the Myo19 membrane-binding motif, we studied the binding of a synthetic peptide representing Myo19 residues 851–895 (Table S2) to small unilamellar lipid vesicles (SUVs) with a phospholipid composition similar to the OMM [55% phosphatidylcholine, 30% phosphatidylethanolamine, 13% phosphatidylinositol and 2% dioleoyl phosphatidylserine (de Kroon et al., 1997)]. Binding was followed by fluorescence anisotropy, relying on two tryptophan residues that are present in the peptide. The binding of Myo19 peptide to vesicles resulted in both an increase in the anisotropy and enhanced fluorescence signal (Fig. 8D). Equilibrium binding of Myo19 peptide to vesicles was monitored by titrating peptide versus increasing vesicle concentration. This resulted in a hyperbolic binding curve of both the fluorescence anisotropy and the fluorescence total intensity (FTI) (Fig. 8D). Therefore, to account for the FTI change during the fluorescence anisotropy measurements, we applied global fitting to both signals arising from peptide binding to vesicles to compensate for the change in fluorescence yield during fluorescence anisotropy measurements. Globally fitting the data to a hyperbolic binding equation (see Eqn 3 in the Materials and Methods) yielded an apparent binding constant of KD=281±50 μM (mean±s.d., n=3). To verify our results that endogenous Myo19 cannot be released from the OMM by high salt in vitro, we examined the salt-dependence of the binding interaction between the Myo19 peptide and the vesicles. As expected, equilibrium binding of Myo19 peptide to vesicles as a function of [NaCl] up to 1 M exhibited no effect on the magnitude of binding (Fig. 8E, inset, slope≈0), supporting our above findings (Fig. 7D). We performed additional binding experiments using a shorter peptide derived from Myo19 membrane motif that contains the peak in predicted helix propensity and the corresponding ER mutant (Fig. S4D, Table S2, Myo19858-883 and Myo19858-RK882/3SS, respectively) to either OMM or ER mimicking vesicles [20% phosphatidylethanolamine, 66% phosphatidylcholine, 9% phosphatidylinositol, 3% dioleoyl phosphatidylserine (Watanabe et al., 1996)]. Both peptides showed similar affinities to OMM-mimicking vesicles (45.0±14.1 and 49.0±17.2 μM for WT or mutant peptide, respectively, Fig. 8F; mean±s.d., n=2). The smaller Myo19-derived peptide exhibited tighter affinity than the longer one. This observation might reflect the predicted higher specificity that resides in the shorter peptide than the longer peptide. The WT peptide had also a similar affinity to ER-mimicking vesicles (50.5±22.0 μM), not showing evident preference for OMM-mimicking vesicles. In vitro binding of a hydrophilic peptide (Zeta1, Table S2), was much weaker (∼2.5 fold) compared to the Myo19 peptide (124.4±18.7 μM), supporting the notion that Myo19–OMM binding is mediated by hydrophobic interactions (Fig. 8F). Although our newly identified membrane motif is essential and sufficient for OMM localization in cells, the cellular specificity might be achieved by unique, yet unidentified, cytosolic or mitochondrial components.
Mitochondria are a key regulator of many stress conditions that cells might encounter, such as deprivation of various nutrients. To survive, cells must possess mechanisms that enable them to regain homeostasis. We therefore focused our investigation on the function of Myo19 in the cellular response to stress by glucose starvation, a major metabolite that is key to many cellular signaling pathways and a stress condition that occurs in solid tumors (Acker and Plate, 2002; Schroeder et al., 2005; Tredan et al., 2007; Hirayama et al., 2009; Moruno et al., 2012). We demonstrate a new function for Myo19 in filopodia formation and mitochondria localization under the cellular glucose-starvation stress response.
Glucose starvation of U2OS cells ectopically expressing Myo19 resulted in striking localization of Myo19 and mitochondria to tips of newly formed starvation-induced filopodia in a motor-dependent manner. Interestingly, these experiments demonstrated a direct link between active Myo19 and the actin cytoskeleton for their localization to the tips of starvation-induced filopodia. Knockdown of Myo19 by siRNA revealed its contribution to the formation and elongation of these filopodia, strongly supporting the hypothesis that Myo19 plays a role in starvation-induced filopodia formation. Finally, we show the molecular details of Myo19–mitochondria interactions, which is achieved through a unique ∼30–45-residue motif that enables it to be directly anchored to the OMM. To the best of our knowledge, this is the first myosin that is anchored in the membrane, in contrast to other myosins, which interact with phospholipids through electrostatic interactions (Hokanson and Ostap, 2006; Spudich et al., 2007). This distinctive feature most likely facilitates strong force bearing ability, preventing detachment of Myo19 from the mitochondria and overcoming high drag forces and competing interactions.
Filopodia are actin-based protrusions that extend from the lamellipodia and have been shown to be important in cancer cell migration, metastasis and angiogenesis (Folkman, 2002; Machesky, 2008; Shibue et al., 2012). Filopodia formation depends primarily on ATP-actin polymerization at the growing tip, providing the energy to push the membrane forward (Mallavarapu and Mitchison, 1999). Molecular modeling performed by Papoian and coworkers (Lan and Papoian, 2008) has modeled the rate of filopodia growth as being dependent on three factors: the resistance of the cell membrane, the mechanical buckling forces that lead to breakage of filopodia filaments, and the transport of ATP-actin monomers to the growing tips. The model also predicts a length-dependence of ATP-actin diffusion from the lamellipodium to the filopodia tips, where eventually the polymerization of actin will be limited by ATP-actin concentration. Ectopically expressed class III myosins have been shown to localize to tips of stereocilia, transporting espin-1, which results in a boost of their growth (Salles et al., 2009; Merritt et al., 2012). Myosin X has been found to undergo intrafilopodial motility and function in filopodia formation (Berg and Cheney, 2002; Tokuo and Ikebe, 2004; Bohil et al., 2006).
We propose that Myo19 localization to the fast growing ends of the filopodia promote their growth probably by mediating the localization of mitochondria to supply the high local ATP demand for ATP-actin polymerization. Here, we noticed that not all filopodia (<20%) contain mitochondria, and we speculate that there are small mitochondria at the filopodia tips, which are below our detection limit. Another possibility is that not all filopodia are being actively elongated or fail to continue to mature. In support of this, it has been shown that mitochondria stalling at the axon branching sites are necessary for efficient branching, an actin-polymerization-dependent process (Spillane et al., 2013). However, we cannot exclude a role for mitochondria in filopodial Ca2+ regulation (Davenport et al., 1996) or other signaling events leading to filopodia formation.
Glucose starvation increases the cellular AMP:ATP ratio, triggering the activation of AMP-activated protein kinase (AMPK) to meet the energetic demand of the cell (Hardie et al., 2012). We found that the minimal glucose concentration that prevents U2OS starvation-induced filopodia is extremely low. Complete medium contains 25 mM glucose, whereas supplementation of the starvation medium with as little as 5 µM prevents formation of starvation-induced filopodia (Table S3). This concentration is much lower than necessary for glycolysis (Faulkner and Jones, 1978); therefore, we propose that the prevention of Myo19–mitochondria localization to starvation-induced filopodia is mediated by glucose signaling events rather than metabolic homeostasis. In support of this, glucose starvation results in activation of a unique signature of phosphotyrosine signaling associated with focal adhesions, actin remodeling and F-actin stabilization (Arber et al., 1998; Miki et al., 1998; Graham et al., 2012). Glucose starvation increases reactive oxygen species (ROS) production (Diaz et al., 2009; Weaver, 2009; Graham et al., 2012; Taulet et al., 2012; Olguin-Albuerne and Moran, 2015). ROS have been shown to be essential for the formation of actin protrusions and well-characterized invadopodia, which have been linked to increased cancer invasiveness (Diaz et al., 2009; Weaver, 2009; Graham et al., 2012; Taulet et al., 2012; Olguin-Albuerne and Moran, 2015). Therefore, it could well be that the effect of Myo19 on starvation-induced filopodia formation is regulated through ROS. It will be exciting to study whether glucose starvation and oxidative stress induces Myo19 post-transcriptional modifications (PTMs), such as phosphorylation, that might modulate its motor activity. Here, we found that, under complete medium growth conditions, ectopic expression of Myo19 causes mitochondria to form globular structures. Previous reports have indicated that ectopic expression of Myo19 affects mitochondrial morphology, causing single mitochondria to obtain tadpole morphology; however, such globular structures have not previously been reported (Quintero et al., 2009). Our analysis shows that these globular structures are in fact clumped mitochondria, rather than hyperfused mitochondria; otherwise loss of MitoTracker intensity would be throughout the entire globular structure (Fig. S1E). This indicates that Myo19 does not promote fusion of mitochondria; however its effect on mitochondrial fission and fusion has not yet been determined. Myo19 knockdown did not cause any obvious morphological changes in the tubular mitochondria network. This inclines that Myo19 is not essential for tubular network morphology; however, we cannot exclude that it might also play some role in intrinsic mitochondrial dynamics or that the reduced levels are still sufficient to maintain the mitochondrial morphology. Corollary to our findings, knockdown of Myo19 has been shown to be important for correct segregation of mitochondria in a subset of dividing cells, whereas the rest were correctly dividing (Rohn et al., 2014).
The nature of Myo19 interaction with the mitochondria is essential for understanding the linkage between mitochondria, Myo19 and the actin cytoskeleton. We demonstrate that Myo19 residues 860–890 are essential and sufficient to cause it to localize to the OMM in cells. This region is moderately hydrophobic, with flanking positive residues, which are essential for correct mitochondrial localization. This targeting system has been shown in several tail-anchored proteins. Tail-anchored proteins have a membrane-targeting domain in their last 30 residues, which is nested in the ribosome when the protein completes its translation, whereas the Myo19 membrane-targeting domain is followed by ∼80 residues. The precise targeting mechanism of Myo19 to the OMM still remains to be determined. Myo19 might compete between the ER and OMM membranes, between targeting proteins such as Asna1/TRC40 and a yet unidentified OMM targeting protein, or transported through the ER to the OMM. Myo19 membrane interaction is highly stable as deduced from our biochemical extractions of endogenous Myo19 and from our binding experiments of the OMM binding motif to vesicles. In addition to the increase in fluorescence anisotropy, we observed strong tryptophan fluorescence increase upon binding of peptide containing the OMM-binding motif to vesicles. Tryptophan fluorescence increase is associated with a shift of the residue from a hydrophilic to hydrophobic environment, suggesting some degree of membrane insertion of the OMM-binding motif (Tsai, 2006). We propose with high likelihood that Myo19 is permanently bound to the OMM through its entire lifetime. This implicates that the regulation of Myo19 activity is most likely performed by a mechanism that modulates its ATPase activity while it is anchored to the OMM. It will be fascinating in the future to study the mechanism by which Myo19 localizes to the OMM from the moment of its translation as it anchors to the membrane. Myo19 enzymatic adaptation has not yet been characterized. It will be crucial to study its mechanochemical properties and how they relate to its cellular function (De La Cruz and Ostap, 2004; Geeves et al., 2005; Henn and Sadot, 2014).
Myo19 has recently been implicated in gliomagenesis (Jalali et al., 2015). Moreover, mitochondria were found in filopodia of glioblastoma cells that actively penetrate extracellular matrix (Arismendi-Morillo et al., 2012). Taken together with our observation that knockdown of Myo19 attenuates filopodia formation without evident effects on the mitochondrial network organization, we propose that Myo19 might be a therapeutic candidate for cancer therapy.
All chemicals, reagents and media were purchased from Sigma (St Louis, MO) unless otherwise indicated.
Myo19 cloning and plasmids
Myo19 was cloned from an EST library (NIH clone ID 3629870) cDNA. The amplified PCR product was missing the region encompassing nucleotides 1318–1916 (database sequence FLJ22865). Therefore, this region was artificially synthesized and cloned into pFC14K (Promega). Full-length Myo19 and its tail domain (residues 824–970) were subcloned to peGFP-N1b using restriction enzymes and used as a template for truncations and mutagenesis. Table S4 contains the primers used for the cloning. peGFP-N1 was a gift from Nabieh Ayoub (Technion Israel Institute of Technology, Haifa, Isreal), fascin–eGFP was a gift from Alexander Bershadsky (Weizmann Institute of Science, Rehovot, Isreal). mEmerald–Vinculin-N-21 was a gift from Michael Davidson (Addgene plasmid number 54304) (Burnette et al., 2011). pRK-GFP-Paxillin was a gift from Kenneth Yamada (Addgene plasmid number 50529). eGFP-tagged full-length mDia2/Drf3 was a kind gift from Klemens Rottner and Jan Faix (TU Braunschweig and Hanover Medical School, Germany, respectively).
Cell culture, cell lines, starvation conditions and cytoskeleton interfering drugs
U2OS cells (gift of Nabieh Ayoub) were grown at 37°C and under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS, Biological Industries, Beit Haemk, Israel), 4.5 g/l glucose, 2 mM L-Glutamine, 20 mM HEPES-KOH pH 7.4, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B. HEK293SF-3F6 (ATCC) for purification of mitochondria and cell organelles were grown in suspension in EX-CELL 293 (Sigma) at 37°C and 5% CO2. For starvation conditions, cells were rinsed twice with PBS and incubated in starvation medium (glucose-free DMEM supplemented with 20 mM HEPES-KOH pH 7.4, 5 mg/ml BSA, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B) for 2 h. Drugs that interfered with the cytoskeleton (final concentration: 0.2 µM Latrunculin B or 0.75 µg/ml nocodazole) were first diluted in medium and then added to the cells. Cells were tested for mycoplasma contamination and found clean (EZ-PCR mycoplasma detection kit, Biological Industries).
Purification of mitochondria
HEK293SF-3F6 cells were harvested by centrifugation at 200 g, washed twice with PBS and once in homogenization medium (0.25 M sucrose, 1 mM EDTA, 20 mM HEPES pH 7.4). The pellet was resuspended in homogenization medium containing protease inhibitors (0.1 mM benzamidine, 0.055 mM phenanthroline, 1 mM PMSF and, from AG Scientific, 0.01 mM bestatin, 0.02 mM leupeptin and 0.005 mM pepstatin A), homogenized using a teflon-glass pestle and centrifuged at 1000 g to pellet nuclei. The resulting post-nuclear supernatant was centrifuged for 20 min at 10,000 g to pellet mitochondria and obtain a heavy mitochondrial fraction (HMF). The supernatant from the 10,000 g centrifugation was taken and centrifuged for 20 min at 17,000 g to pellet light membranes and obtain a light mitochondrial fraction (LMF). The supernatant from the 17,000 g was taken to obtain a cytosolic fraction (CYT).
Additional purification of mitochondria
To obtain pure mitochondria we used isopycnic ultracentrifugation. The HMF was resuspended in homogenization medium containing protease inhibitors and adjusted to 17.5% OptiPrep. The HMF was centrifuged at 270,000 g for 3 h in a Sorvall T-890 or Beckman Type 70 rotor to form a self-forming density gradient. Following ultracentrifugation, three bands appeared: light membranes, mitochondria and a DNA-rich band (from top to bottom). The second band was taken using a pasture pipette, diluted 1:1 with homogenization medium containing protease inhibitors and centrifuged for 30 min at 17,000 g to pellet mitochondria. The mitochondria were resuspended in fresh homogenization medium containing protease inhibitors at ∼5–6 mg/ml, concentration was measured using a Bradford assay according to the manufacturer's directions (BioRad). Purified mitochondria were flash frozen in liquid nitrogen and stored at −80°C. All steps were performed at 4°C.
Protease protection assay
For proteinase K digestion, mitochondria were washed three times with homogenization medium to remove protease inhibitors. Proteinase K was added at 50 µg/ml and incubated on ice for 30 min. Reactions were terminated by addition of PMSF to 2 mM and centrifugation at 10,000 g for 3 min to pellet mitochondria and to remove the protease. Sample buffer ×5 (final concentration, 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% Bromophenol Blue) was added to the samples and they were resolved by SDS-PAGE.
Membrane extraction
Purified mitochondria were resuspended in either 100 mM NaCO3 pH 11.5, 2 M NaCl in homogenization medium, or 2 M urea in 20 mM MES pH 6.5 for 30 min on ice, during which the NaCl and urea samples were subjected to ten freeze–thaw cycles in liquid nitrogen to mechanically break the mitochondria. The samples were then centrifuged for 1 h at 150,000 g in a Sorvall S100-AT3 rotor. The supernatant was collected and the pellet was resuspended in an equal volume of 100 mM HEPES pH 7.4. To precipitate the extracted proteins, we performed trichloroacetic acid (TCA) and acetone precipitation on the NaCO3 and NaCl supernatant, TCA was added to 12% from a 100% stock and allowed to incubate for 1 h at 4°C. The samples were centrifuged for 30 min at 17,000 g, resuspended with ice-cold acetone and left overnight at −20°C. The samples were then centrifuged for 30 min at 17,000 g, supernatant was removed and the acetone was allowed to evaporate by heating the samples for 10 min at 90°C. Urea-containing samples were desalted using Zeba spin desalting columns 7K MWCO according to the manufacturer's protocol (Thermo Fisher Scientific). Sample buffer ×5 (as described in the protease protection assay section) was added to the samples and they were resolved by SDS-PAGE.
Triton X-114 extraction
Purified mitochondria were resuspended in cold phase separation buffer [10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-114 (MP Biomedicals); protease inhibitors as described in the purification of mitochondria section] and incubated on ice for 5 min, insoluble material was removed by centrifugation at 10,000 g for 10 min. The lysate was loaded on top of a cold sucrose cushion (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.06% Triton X-114, 6% sucrose, protease inhibitors), incubated at 37°C for 3 min and then centrifuged at 300 g for 3 min. The upper phase was placed in a new tube and Triton X-114 was added to 0.5%; the solution was then incubated on ice until it cleared, loaded on the sucrose cushion, incubated at 37°C for 3 min and then centrifuged at 300 g for 3 min. This process was repeated again with the exception that Triton X-114 was added to 2%. The upper and the lower phase were collected and corrected to the same volume. Sample buffer ×5 (described in the protease protection assay section) was added to the samples and they were resolved by SDS-PAGE.
Western blotting
Following SDS-PAGE, gels were washed in Milli-Q water. Nitrocellulose or PVDF membranes (PVDF membranes were hydrated in 100% methanol for 1 min) were incubated for 1 min in transfer buffer and transfer was performed for 10 min using a semi-dry blotter (GenScript). Transferred membranes were washed with Milli-Q water and blocked using EZblock (Biological Industries) for 1 h at room temperature. Following blocking, primary antibody was added for 1 h at room temperature or left overnight at 4°C [0.5–1 µg/ml anti-VDAC antibody abcam ab14734, anti-Myo19 antibody Sigma HPA021415 or Abcam ab174286, anti-GRP94 antibody (a kind gift from Christopher Nicchitta, Department of Cell Biology and Biochemistry, Duke University, North Carolina), anti-HSP60 antibody (Santa Cruz Biotechnology sc-59567), anti-ENDO G antibody (Santa Cruz Biotechnology sc26923), anti-ATP5A antibody (abcam ab14748) anti-GFP antibody (Roche 11814460001)]. The membrane was washed with TBS-T (TBS, 50 mM Tris pH 7.5, 150 mM NaCl with 0.02% Tween-20) and horseradish peroxidase (HRP)-conjugated secondary antibody was added for 1 h, washed with TBS-T and developed using 0.01% H2O2, 1.25 mM Luminol and 0.04 mM p-Cumaric acid in 0.1 M Tris-HCl pH 8.5. Images were acquired using a ImageQuant LAS4000 imager (GE).
Transfections and microscopy
Transfections were performed using polyethylenimine (PEI, Weis Scientific-PolySciences). Adherent U2OS cells were plated a day before transfection on plastic or glass bottom dishes and allowed to adhere overnight. Plasmid DNA and PEI were diluted separately in 150 mM NaCl and then combined, and complex formation was allowed to occur for 25 min at room temperature before addition to the cells and incubation overnight. Hoechst 33342 (0.75 µg/ml), MitoTracker (30 nM, Molecular Probes) and propidium iodide (1 µg/ml) were added 15 min prior to imaging. HaloTag constructs were stained by incubating the cells with HaloTag TMR Ligand ON (25 nM, Promega). Cells were imaged using Confocal Zeiss LSM 700 or Confocal Zeiss LSM 710 or GE InCell Analyzer 2000 at ×63 magnification in an environmental chamber.
Cells grown on coverslips were washed with PBS, and fixed with 4% PFA in PBS (EMS) at room temperature for 15 min. The PFA was removed by washing with PBS, and the cells were blocked for 1 h with EZblock (Biological Industries). To visualize actin and nuclei, the cells were incubated with Alexa-Fluor-488–phalloidin and Hoechst 33342 for 30 min. The cells were then washed with TBS and mounted on slides using Fluoromount-G.
RNA interference
Transfections were performed using Lipofectamine 3000 (Life Technologies). Adherent U2OS cells were plated at 70–90% confluence a day before transfection on plastic dishes and allowed to adhere overnight. Mock siRNA (Universal negative control siRNA, IDT) or mix of three Myo19 siRNAs (10 nM each, reference numbers 118568291, 118568285, 118568288, IDT), and Lipofectamine were separately resuspended in OptiMEM (Life Technologies). The RNAi mix was added to the Lipofectamine mix and incubated for 5 min at room temperature. The medium was replaced with DMEM without serum and the transfection mix was added. Complete medium was added after 4 h. On the next day, the cells were trypsinized and plated on glass bottom fluoridish (WPI) or optic bottom 24-well plates, for microscopy, or plastic dishes, for western blotting at 72 h post transfection.
Prediction webservers used in this study
The webservers can be found at http://heliquest.ipmc.cnrs.fr/, http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/, http://www.sbc.su.se/~miklos/DAS/.
Image analysis and calculation of mitochondria-positive Myo19–eGFP foci
Western blot band intensity, filopodia length measurements and line plot analysis were performed using ImageJ (NIH). Filopodia angle measurements were performed using IMARIS V8.0.0 (Bitplane AG, Zurich, Switzerland).
To calculate the fraction (f) of mitochondria positive Myo19-eGFP foci (Fd) we calculated the ratio between the number of MitoTracker-Red-positive Myo19–eGFP foci (NFd) to the total number of Myo19–eGFP foci (NFt) according to the equation:
Determination of filopodia growth rate
To determine the filopodia growth rate from the time-lapse imaging, we took into consideration only the region where the filopodia growth was constant over time (i.e. between 80 and 110 min). We used the following equation to calculate individual filopodia growth rate vf: where l110 is filopodia length at the time of 110 min, l80 is filopodia length at the time of 80 min and elapsed time (Δt) is 30 min. N=30 for each treatment.
SUV preparation
Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and dioleoyl phosphatidylserine (Avanti) were mixed in chloroform and evaporated under nitrogen. The vesicles were then resuspended in binding buffer (40 mM HEPES pH 7.1, 150 mM NaCl), subjected to ten freeze–thaw cycles in liquid nitrogen and kept at −80°C. The vesicles were thawed and sonicated for 5 min at low intensity; insoluble material was pelleted by centrifugation at 100,000 g for 20 min. Vesicles were used within 2 days after sonication.
Microirradiation of mitochondria
U2OS cells were loaded with 25 nM of the MitoTracker dye (sensitive to mitochondrial membrane potential) for 15 min at 37°C. Unbound dye was removed by three washes with complete medium. A 0.5×0.5 µm square was irradiated using a 405 nm laser at 100% intensity, causing single mitochondria to depolarize and lose staining.
Fluorescence anisotropy measurements and KD determination
Equilibrium binding by fluorescence anisotropy measurements was performed with a PC1 spectrofluorimeter (ISS, Champaign, IL) designed as a T-format for simultaneous acquisition on two emission channel monochromators equipped with automatic polarizers. Samples were equilibrated (60 min, room temperature) and then measurements were performed by following intrinsic fluorescence of the peptide with λex=280 nm using vertical polarized light, and the emitted vertical and horizontal polarized light was monitored at 90° with double emission monochromators at λem=335 nm. The G-factor for correction of the different gain between the dual PMT detectors was calculated as described by the instrument manufacturer. The fluorescence total intensity (FTI) and total fluorescence anisotropy are given by: (1)and (2)where the I|| is the parallel fluorescence intensity and I⊥ is the perpendicular fluorescence intensity.
Our binding model for a simple bimolecular reaction is: Under the condition of Ptot≪KD then the general solution for this equilibrium binding scheme is in the form of the following quadratic equation: (3)where Ptot is monitoring species; Vtot is titrating species and [PV] is the bound species.
The total fluorescence and anisotropy were fitted globally (Origin Lab 9.0) according to Otto et al. (1994) and Henn et al. (2010).
Statistical analysis
Statistical significance between mock and treated groups was performed using Student's t-test, the different groups were considered to have the same variance because the groups were taken from the same pool of cells. Statistical significance was scored as *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.001. All presented error bars indicate standard deviation.
We acknowledge Dr Dan Cassel for his assistance in the vesicle preparation, and critically reading the manuscript. We thank Drs Yehuda G. Assaraf and Nabieh Ayoub for their invaluable comments on the manuscript. We are grateful for Dr Nitzan Dahan's assistance with the fluorescence microscopy imaging from Life Sciences and Engineering Infrastructure Unit at the Technion. We thank Drs Michael Davidson and Kenneth Yamada for providing the focal adhesion marker plasmids. We thank Drs Klemens Rottner and Jan Faix (TU Braunschweig and Hanover Medical School, Germany, respectively) for providing the eGFP-tagged full-length mDia2/Drf3 plasmid.
Competing interests
The authors declare no competing or financial interests.
Author contributions
B.I.S. designed and carried out experiments, analyzed the data and co-wrote the manuscript. M.U. designed and carried out experiments and analyzed the data. A.H. supervised the project, designed and analyzed the data and co-wrote the manuscript.
M.U. is supported by the Lady Davis Fellowship and Israeli Government Ministry of Foreign Affairs Scholarship. A.H. acknowledges the Marie Curie Career Integration [grant number 1403705/11].
Supplementary information
Supplementary information available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.175349/-/DC1
- Received June 9, 2015.
- Accepted December 5, 2015.
- © 2016. Published by The Company of Biologists Ltd


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Online Tour: Mendel Room
Slocum Room | Reading Room | Portrait Gallery | Mendel Room | Wordsworth Room
This room is named for Bernardo Mendel, whose extensive personal library of books and manuscripts on European expansion into the Western hemisphere, particularly the Spanish empire, came to Indiana University in stages between 1961 and 1967. A small selection of his books is kept in this room. The room is furnished in the style of late fifteenth-century Spain.
Books were an avocation in Mr. Mendel's early years, but they became a full-time occupation with him in the later years of his life. He followed with the greatest interest the book trade in Europe and the Americas up until the day of his death. He thought of books as the chief of his legacies to posterity, and his last years were spent enlarging, rounding out, and perfecting his collections.
Bernardo Mendel also had a lifelong interest in music. Periodically, from 1961 to the end of his life, he made gifts to the Lilly Library of selected works from his extensive private collection of music, both orchestra and piano scores. After his death the Lilly Library's collections were further enriched with rare editions of Beethoven, Chopin, Liszt, Mozart, Schubert, Schumann, Strauss, Richard Wagner, Weber, and many other composers. Mr. Mendel had purchased the collection of sheet music formed by the late Dr. Saul Starr, of Eastchester, New York and in 1970 the Starr sheet music collection was given to the Lilly Library. It consists of more than 100,000 items covering almost 300 years of national, folk, and popular American music.
The books and manuscripts which Bernardo Mendel assembled, now in the Lilly Library, will serve and inspire future generations of scholars, teachers, and students.
Above: Portrait of Bernardo Mendel, 1895-1967
photograph © Dave Repp, 1997.


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How Much Would You Pay To Fill Your TV Screen With Only The Images You Want?
By Joanna Robinson | Pajiba Love | March 20, 2012 |
By Joanna Robinson | Pajiba Love | March 20, 2012 |
I'm hoping this story will wrap your cold bitter hearts up in a cuddly blanket. In a rad thread on Reddit, a woman who will turn 100 years old in two days is answering questions. If this is some kind of hoax, I swear I will burn the internet down. Thanks, mswas! (Reddit)
Speaking of hoaxes, you may have heard something about the Mike Daisey/NPR/lying liar scandal. If you missed it, one of Daisey's monologues was aired on "This American Life" as "news" when it was, in fact, largely fabricated/exaggerated. Having seen a few of Daisey's monologues live, I'm not sure how they could ever be mistaken for the truth, but, at the same time, I'm shocked SHOCKED that anyone would lie to Ira Glass. (Washington Post)
For those of you still following, TechCrunch has an interview with the reporter who "exposed" Daisey's lies. (TechCrunch)
If that fails to "shock" you (and it does me, this isn't Stephen Glass redux, no matter what people say), then maybe these six shocking moments from mainstream TV will. I never saw this episode of "Punky Brewster," I think I would have been scarred for life. Obviously, spoilers abound. (Unreality)
Naturally, one of those shocking moments on that list came from the last season of "Game Of Thrones." How excited are we for season two, my little Wildlings and White Walkers? I'm so excited I'm willing to include this new sketch of a rather old joke.
Speaking of Ye Olde Dramaes, the BBC has hired Matthew Macfadyen (the lesser Darcy) for a new Victorian crime series called "Ripper Street." Personally, I'm still waiting for the long ago promised Anthony Stewart Head vehicle "Ripper." (Deadline)
I've got two little memes for you this morning, pets. The first is this more-entertaining-than-it-should-be "One Tiny Hand" meme. Someone on Twitter mentioned "Tiny Handjobs" and I nearly snorted my delicious French Roast. (Uproxx)
The second meme is significantly weirder (I know, the internet, she is strange). I present, celebrities who look like mattresses. Oh, January Jones, I can't wait for "Mad Men" to return so I can resume hating you. (Facebook)
Speaking of Tiny Handjobs, Esquire has published their 2012 Sex Survey. Did you know that 10% of men don't believe Oral Sex counts as Sex? DESPITE IT HAVING THE WORD SEX IN IT? Oh, Bill Clinton, what hath you wrought? (Esquire)
Speaking of Clintons, apparently Hillary is spearheading an investigation into what happened to Amelia Earheart. Why Hillary? Click the link and you'll see. (LaughterKey)
There's a new batch of stills from The Avengers where everyone looks grim and upset. Obviously, these images come from that latter half of the movie after Joss has murdered everyone's favorite character. I will say this, Jeremy Renner gives good glower. (DailyBlam)
I want to snuggle all of these nerd mash-ups. I'm not sold on the title "I Know That Feel Bro." Dear nerds, you're supposed to like English. . .please use it. (Nerd Approved)
Oh you thought that was nerdy? HA! That is but a drop in the nerd bucket. This essay on the efficacy of the Death Star trash compacter, however, is full nerd. It's gone plaid. (McSweeney's)
It may be maudlin, but that's not going to stop me from following this Titanic Twitter account. In honor of the 100th Anniversary, someone is tweeting as if they were the captain and crew. Things are going to get real interesting in about 26 days. (Neatorama)
I found this article on the way in which streaming music sites (e.g. Spotify) are affecting the Billboard charts. Also, I'm super ashamed to admit that I like that "We Are Young" song and was even more ashamed when I found out the band's stupid name. (Village Voice)
Some teacher has been suspended for reading "pornographic material" to his kids. That material? "Ender's Game." Mmmmm, that ol' time religion strikes again. Also, if you think that is disturbing, check out the local media coverage of the teacher's suspension. (io9)
Dustin has a depressing look at how much you could be paying for cable if channels were offered a la carte. That's right, all classy cleavage all the time. That's the dream, man. (Warming Glow)
One of the best things on my TV, Aziz Asari, has elected to go the Louis CK route and distribute his new stand-up special himself. You can buy it on his website for only $5. RIGHT NOW. (Aziz Ansari)
Speaking of "Parks and Recreation" stars, the lovely Chris Pratt proves he's more than just a lovable idiot. He's a lovable idiot in a Jedi robe.
Finally, the very best political parody I've seen so far this campaign season. Won't the real Mitt Romney please stand up?


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Jump to navigation
The latest international news, analysis and features on the HIV epidemic from Avert. Share your views and expertise with your peers in the comments box below the articles.
New research from Lesotho provides more insight into what types of community-based HIV testing and counselling (HTC) are most effective in reaching people with HIV services in resource-limited settings. The cluster randomised control trial, published in PLOS One, looked at whether home-based HTC (HB-HTC) resulted in a higher uptake of HTC than delivery through community gathering and mobile clinics (MC-HTC).
The number of babies born with HIV in Latin America and the Caribbean has decreased by 78 percent – from 10,700 in 2001, to 2,324 in 2013 – according to a new report by the Pan American Health Organization/World Health Organization (PAHO/WHO), and the United Nations Children's Fund (UNICEF). The report entitled, Elimination of Mother-to-Child Transmission of HIV and Congenital Syphilis in the Americas, documents progress made towards the 2015 goals of eliminating mother-to-child transmission (MTCT) of HIV and congenital syphilis in the region.
Women living with HIV on antiretroviral treatment (ART) are more likely to report single and dual-method contraception use, the longer they are enrolled on HIV treatment. The findings, reported in JAIDS this week, looked at research from a rural community in KwaZulu-Natal, South Africa, and is the first of its kind to look at uptake of contraception in women as they progress along the HIV treatment cascade.
With the passing of International Human Rights Day on 10 December, it is important to recognise the link between HIV and human rights. Stigma and discrimination characterise the global HIV epidemic making it difficult for key populations to reach the HIV prevention and treatment services they need.
Poor gaining of weight in children under the age of 10 on antiretroviral treatment (ART) is associated with an increased risk of mortality. According to a recently published study in AIDS Journal, children with poor weight gain after 6 and 12 months of ART initiation have a higher risk of death in comparison to children who gained good weight.
Delivering positive messaging about safer injecting practices could have a greater impact on engaging people who use drugs in harm reduction programmes.
Our World AIDS Day 2014 blog brings together voices from the field to share perspectives on the HIV epidemic on this day of remembrance and solidarity. This blog series includes messages from AVERT's CEO, our partner, the Umunthu Foundation, and a case study from our South African partner, Sisonke. The blog also features guest pieces from the International HIV/AIDS Alliance, and the Key Correspondents programme.
Dear all young people,


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$35.00 $45.00
Two round Labradorite gems are featured on each end of the Sterling Silver bypass ring. With tiny silver bits played as pollens, the whole piece is indicative of a flower in bloom. The magnetic play of color in the Labradorite gemstone is associated with the Aurora Borealis and its powers.
Wearing this Astra Labradorite Ring is like parading the Northern Lights in your hands. And as its energies envelope you, you'll feel a blanket of protection that can also amplify your mood.


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St Terese of Lisieux, also known as the Little Flower
Prayer of St Terese of Lisieux
May today there be peace within.
May you trust God that you are exactly where you
are meant to be.
May you not forget the infinite
possibilities that are born of faith.
May you use those gifts that you have received,
and pass on the love that has been given to you.
May you be content knowing you are a child of God.
Let this presence settle into your bones,
and allow your soul the freedom to sing,
dance, praise and love.
It is there for each and every one of us.
I just loved making this shrine of St Terese.
She is known as the Little Flower and is well-known for her
beautiful sayings comparing the inner life to a garden.
"The splendor of the rose and the whiteness of the lily
do not rob the little violet of it's scent nor the daisy of its simple charm.
If every tiny flower wanted to be a rose, spring would lose its loveliness."
do not rob the little violet of it's scent nor the daisy of its simple charm.
If every tiny flower wanted to be a rose, spring would lose its loveliness."
The shrine is filled with flowers and roses
and I covered the wooden cross with a multitude of roses.
I used fabric roses, aluminium roses, paper roses,
porcelain roses and german glass glitter on the roses
on the holy card image of the sacred heart.
St Terese said that after she had gone she will
shower roses from heaven.
The rose is a powerful symbol for many reasons.
It has five petals that shows at any given time,
representing the five elements,
thus representing wholeness.
I used all sterling silver charms on the side;
a brass finding as a halo behind her head;
velvet ribbon and vintage lace on the frames
as well as
hand punched lace paper trim.
I was so happy when I found these punches.
This shrine is part of the Mary Sacred Rosary Oracle Deck -
a huge project that I have undertaken!


text: The "Quick Fit" case is suitable for storing the "Basic I" Horseshoe Lifebuoys (LZT-20530, LZT-20540,LZT-20550). It is made of Polyester fabrec, of 0.33mm thickness, with PVC backing, which makes the case waterproof, protecting the lifebuoys from most weather conditions. The case features a quick Velcro open - close system and straps for mounting the case on your boat's rails. Its design allows you to store the lifebuoy light as well. It is available in white & yellow colour.


text:
The Coach Who Clothed Them - Uncensored - Buxrs.com
Published: 3 years ago By: Comedy Central
By: Comedy CentralPublished: 3 years ago
6, 026, 527 views
54, 907 Likes 3, 377 Dislikes
Steve Rannazzisi stars in a new movie about a man who stands up to an unjust system -- no matter the cost.
The Comedy Central app has full episodes of your favorite shows available now. http://on.cc.com/1e85GN8
Directed By: Brandon Dermer
Written By: Jeff Danis and Ryan O'Neill
Coach: Steve Ranazzissi
Player 1: Amanda Cerny
Player 2: Lizz Adams
Player: Whitney Moore
Player: Christina Elise Escobedo
Player: Molly Mitchell
Player: Laura Ashley
Player: Kijai Furtak
Man 1: Jeff Danis
Man 2: Ryan O'Neill
Guy: Blake Wexler
Woman: Jackie Kashian
Owner: Jonathan Marballi
Commissioner: Daniel Van Kirk
Thug: Carl Tart


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January 24, 2017
Sir, I refer to the so plentiful anti-trade-protectionism writings, in FT and everywhere, and which all warn about the dangers of what Big Bad Donald Trump is up to. Many of these, not all, are solidly argued. Yet these contrast so much with the almost absolute silence against the financial protectionism that is imbedded in current bank regulations.
The risk weighted capital requirements for banks allow banks to leverage more with assets perceived, decreed or concocted as safe, than with assets perceived as risky. That means banks will earn higher risk adjusted returns on equity on the "safe" than on the "risky; so banks will favor with credit or investments what's safe over what's risky.
Is this not how it always is. Yes but before the introduction of these capital requirements the perceived risk was cleared for by the size of the exposure and the risk premiums charged. Now when capital requirements are also based on the same perceived risks, the effect of these in the allocation of bank credit to the real economy are augmented and so distort.
Here are some risk-weights of Basel II. Sovereign=0%, AAArisktocracy=20%, residential housing=35%, not rated "We the People", like SMEs=100%, below BB-rated=150%.
Those who do not see how those with lower risk weights have their access to bank credit protected, against that of the risky, are not interested, dumb or trapped, almost irreversibly, by the mother of all confirmation biases.


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Grant County High Point Trip Report
one area on the county line (3,220+ ft)
Date: January 2, 1998
Author: Dave Olson Conditions were very sloppy, muddy, snowy. I canceled plans to visit the Stevens county HP areas and went to the HP area for Grant county KS instead. That was another undistinguished roadside pasture. The interesting part was finding my way afterwards back to civilization to the north.


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Tuesday, August 19, 2008
Why People Become Supervillains
I'm really very sorry. I couldn't resist doing this to this panel. But, hey, who's to say Sinestro isn't a victim of spurned affections? After all, darned near every other Green Lantern supervillain is in love with Hal.
Posted by Sea-of-Green at 10:27 AM
Labels: Green Lantern, Hal Jordan, Making fun of GL, Sinestro
4 comments:
- Riddering said...
Sinestro sure falls fast, doesn't he? One mention of doubting the yellow impurity and the next thing you know he's ready to indulge in some very disorderly activities!
- August 19, 2008 at 4:38 PM
- ComicsAllTooReal's Chris said...
That explains it all. Sinestro isn't the loving type, that could be Hal's fault. Poor Sinestro, so broken hearted.
- August 20, 2008 at 1:08 AM
- SallyP said...
Sea, I think that you've hit the proverbial nail on the proverbial head.
- August 20, 2008 at 10:40 AM
- Sea_of_Green said...
I'm sorry, but it was the first thing that popped into my head when I first saw this panel. THAT'S IT! SINESTRO LOVES HAL!
That's GOTTA be it ...!
- August 20, 2008 at 3:00 PM
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