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Artisan Countries
With a mission to restore hope to exploited women in Asia, Starfish Project established a socially responsible jewelry company in 2006 as a means to offering meaningful alternative employment, vocational training and educational grants, and a wide array of social programs to women trapped in exploitative situations. Each woman who leaves an exploitative situation for employment at Starfish Project is given opportunities to heal her physical and emotional scars in ways that transform every aspect of her being, including in her relationships with family, friends, and colleagues. By operating under three main values—the holistic transformation of lives, implementing ethically based business and organizational practices, and raising up local leadership—Starfish Project continues toward a vision of seeing every woman valued. Each piece of jewelry purchased directly funds the empowerment of exploited women in Asia.
The thin, dirty girl sits dejectedly on the curb as pedestrians filter past her. Ten-year-old Farjana is tired and does not feel well, but she knows she must earn more money before she can go home for the day. "When I was very young, my father died," Farjana says. "My mom was unable to manage our family by providing food and other needs for us. . . . The last 6 years I have been going to [a major intersection] for begging and earning money for my family."
Farjana does not go to school and cannot read or write. She wants to go to school someday because "without education, there is no significance in life," she said.
She is just one of Bangladesh's estimated 700,000 beggars. Girls from low-income families like Farjana face many obstacles and dangers in their life—including potentially being trafficked and exploited, says worker Geri Hennerman. "They are seen as nobodies and only good for cleaning, cooking, sex, and child-bearing," she says. "They are often beaten verbally and physically. They have little—if any—encouragement, love, correct discipline, or teaching. They do not have good female role models to follow. Many are given for marriage at extremely young ages. They are seen as objects instead of real people."
But the Light of Hope Learning Center, which Geri started more than 6 years ago, helps prevent impoverished girls from having this dark future. The center is a day shelter that gives girls education, life skills, health care, and moral training. The girls learn they are special and have great potential for living a transformed life. "They spend their days learning and growing in all areas—intellectually, morally, spiritually, physically, emotionally, and socially—instead of spending their days (and nights) out in their very dangerous and open areas, learning the ways of the world and getting into dangerous and trouble-type situations," Geri says.
In a small village in southwest Bangladesh, widows frantically search for work in order to take care of their young children. Because the widows are uneducated, they don't possess marketable skills, and this leaves them with a bleak view of their future. But for the last several years, Peaceful Creations has partnered with the widows to sell their handmade items. By creating these items, more than 50 widows are now able to stay at home with their children while earning an income.
Rescue is only the first step on the path to restoration for victims of exploitation and trafficking. Even after extensive therapy and time to heal, former victims are extremely vulnerable to being exploited again if they have no ability to earn a viable income and provide for themselves and their families. Without skills and jobs, the likelihood of being in harm's way again is almost guaranteed.
Sak Saum recognizes this reality and is committed to ensuring that those in their care are equipped with skills and employment and earn fair wages in a safe, loving environment. They also seek to pre-empt exploitation by equipping and hiring the most vulnerable in the local community before they fall prey to traffickers.
Every Sak Saum product is made by someone who now has skills and works with dignity. By purchasing a Sak Saum product, you are ensuring that the work of rescue, restoration, and transformation continues.
Although women of China believe in tradition, they desire to break away from the cycle of poverty, domestic abuse, and the lack of education. For generations, Chinese women have embellished their clothing with extravagant colors and intricate designs, but they only began marketing their talent when they joined China Ethnic Crafts. Now, women can embroider flowers, birds, and butterflies on accessory items. This work earns their families extra income to supplement rice farming and send their children to secondary school.
The Lily House in the Dominican Republic offers healing to women who have been sexually trafficked and those who are at risk of being trafficked. With high levels of poverty, illiteracy, and physical abuse, Dominican women are exceptionally vulnerable to traffickers. The Lily House teaches women to create beautiful jewelry. In the process, these formerly enslaved women learn that they are themselves beautiful creations worthy of love. "I knew how to do nothing with my hands before Lily House," says N. "The only thing I knew to do was sell my body. Now I make beautiful things with dignity." Your purchase of jewelry made by The Lily House artisans brings hope to those who were without hope.
Artisans at Tabitha Ministries in Guatemala make exquisite products out of seed beads. The group employs eight artisans who are able to provide much-needed income for their families through their work.
Maria is an artisan who can pay for school and support her family with the money earned from products sold through WorldCrafts. She is able to purchase things like firewood and corn. She works so hard to provide for herself and family. She is studying business administration and wouldn't be able support her family without her job doing bead work.
Bidri Ware employs skilled artisans in a southern Indian town. The artisans use an ancient handicraft skill that features alloy of zinc and copper inlaid with thin sheets of silver. The eight-stage process entails molding, smoothing, chiseling, engraving, silver inlaying, smoothing (again), buffing, and oxidizing. Through their employment with Bidri Ware, the artisans receive increased payment for their work, which allows them to better provide for their families.
Channapatna Handicrafts is located in a quaint southern Indian town. The artisans use a special skill called lac-turney, a 200-year-old craft traced to the reign of a royal ruler who invited artisans from Persia to train the local artisans in making wooden toys. The process of shaping and coloring the wood is carried out on a lathe, a machine that turns pieces of wood while the artisans use sharp tools to shape them. Artisans then apply a vibrantly colored lacquer made by mixing natural, nontoxic vegetable dyes.
These lac-turney products are carved from hale wood, also called ivory wood, a local species of tree that grows widely throughout south India. Usually the entire tree is not felled, and the tree grows back. Therefore, the materials used in the craft are natural and eco-friendly.
In the last decade, without proper support in marketing, the number of Channapatna artisans has declined considerably. The craft was dying. With help of WorldCrafts, the artisans of Channapatna have been able to reach the global market, helping the sales of handmade products and improving the livelihoods of the artisans and their families. WorldCrafts empowers the artisans by reviving the craft, providing better working conditions and sustainable incomes, and creating employment opportunities in the community.
In cities such as Kolkata (Calcutta), India, many women live in traditional patriarchal communities and are not allowed to work outside their homes. ConneXions offers these women the opportunity to collect the raw materials for their quilts and scarves and take them home to produce. This opportunity to work from home and earn a living wage opens the door to economic empowerment for the artisans. With the income received through their employment with ConneXions, the artisans are able to increase the educational opportunities for their children (especially their daughters) as well as help their families survive setbacks, particularly when illness or unemployment strikes.
Within each stitch of the Himalayan Tapestry artisans' handiwork is a story of illiteracy, abuse within marriage, and culturally conditioned gender discrimination. While these Indian women suffer quietly, they are expected to carry the financial burden of their families. The women come to Himalayan Tapestry with little education and no basic skills, but here they learn to quilt, tailor, read, and manage money. Working with this organization enables women to make money from their new talents, and it enables them to receive crisis counseling, which brings financial and emotional comfort to their troubled lives.
The scarring influence of the sex trade does not affect just one generation in India. Despairing mothers often leave a legacy of poverty and prostitution to their daughters, who in turn carry on because they know no other lifestyle.
Enter Love Calcutta Arts. Their handicrafts unit enables young women to support themselves with dignity and help their mothers leave the sex industry. These hardworking women make high-quality handmade paper products in a loving environment where they learn of their Father's love for them.
Noah's Ark is an innovative network of 100 fair-trade artisan groups, representing 600 individuals, across India. Artisans work with a variety of materials, including wood, paper, and metal, to create contemporary products using traditional methods. All workshops are committed to providing sustainable income in an environment of honesty and integrity. As Noah's Ark grows it has been able to support schools for artisan children and provide filters for clean water.
Mary grew up in an orphanage in India with a bleak outlook for her future. When time came for her to leave the orphanage, she was deceived by an offer of marriage and found herself instead in a brothel. She escaped and tried to commit suicide multiple times, the last time by eating glass. A member of Rahab's Rope artisan group found her dying in the street and brought her to the center. Many young women like Mary have found a lifeline at Rahab's Rope, where they discover new opportunities while making handcrafted items.
The young women of Shelano have come from the slums in the south of Kolkata, India. Finding a job is difficult, if not impossible, with their background and limited education. By purchasing their handmade products, you are not only getting a fairly-traded product, but also giving a woman an opportunity to take care of her family and invest in the future. Each throw is unique and is made by a unique woman with a unique story. As it goes from her hands to yours, you are help a woman earn some money of her own and contribute to her confidence and well-being.
The White Rainbow Project is transforming lives by sharing love with the widows of India. It provides vocational training, food, and medical care to destitute widows in Vrindavan, India. Vrindavan, known as the City of Widows, is home at least 20,000 widows who have been abandoned by their families. White Rainbow Project is recycling hope for these widows by helping them create beautiful handmade products out of donated saris and old magazines. Recycling takes on a whole new meaning, as this is one of the only options widows have to support themselves. They have been told they have "bad karma" and are blamed for the death of their husband. They are shunned, exploited, and denied any sense of dignity. Earning their own money gives them freedom to choose their own destiny, many for the first time in their life.
Impoverished Indonesian mothers struggle with providing basic necessities for their families. Most of their husbands receive a salary of only around $100 a month. These families live from month to month and barely make enough money to feed their families, much less provide for other needs such as schooling.
Ayu Sewing Project was created to help Indonesian women learn a trade that they could use to support their families using their new skills. Originally, they made scarves. By making only a few scarves per week, these mothers are increasing their monthly income by 40%! The women use the money to pay for their children's schooling, medical, and rental expenses.
In the Middle East, people with disabilities are often forced to become "takers," looking for handouts. Glad Tidings provides dignity for its artisans as they earn a living to provide for themselves and their families. When asked about her hope for the future, one artisan shared, "I don't dream about my future. I dream about the future of my kids. I dream that one day they will be able to get a college education!" For the unloved and rejected, Glad Tidings continues to bring hope and love.
Back to Africa began in 2008 as an offshoot of Heart of the Bride Ministries, Inc. The primary objective of Back to Africa is family preservation and care for families at risk in Kenya. The artisans, most of whom are single mothers or refugees or both, lived in extreme poverty before they began creating Back to Africa jewelry.
Mama Faith, pictured here, has been working with Back to Africa since 2008 as a bead artisan. A mother of 4, her income from Back to Africa enables her to consistently feed and clothe her children, send them to school, give to her local church, and help her family and neighbors who are not as fortunate. She says, "I have seen God in these necklaces. My husband got saved and baptized! I am a very happy woman. He used to drink and now he goes to church, and helps with the beads."
The Back to Africa project has benefited more than 400 people. They have seen firsthand how a small paper bead or simple ceramic pendant can profoundly impact a community halfway around the world.
Mother Care Handcrafts helps disadvantaged artisans in Kenya earn a fair wage by using their skills in the centuries-old traditions of carving and basket making. Among the artisans are several women who have been rescued from prostitution, thanks to the jobs Mother Care offers. Sales provide guild artisans a way to feed and clothe their families, as well as affording them an opportunity to hear the offer of eternal life.
Mully Children's Family (MCF) created Yatta Vocational Training Centre to restore young women broken by exploitation. Each year, the training center rescues approximately 100 victims out of this cruel bondage. When they arrive at the training center, these women receive counseling and learn vocational skills, computer training, and family care. They are also discipled in developing lives marked by eternal hope.
Young women like Felister Mwikali are taught beadwork, dress making, and design. Felister came to the center after being abused by a man who lured her out of her village with promises of a better life. However, he threw her out of his home when she became pregnant. Upon returning to her family, she was treated with disdain due to her pregnancy, and she was not allowed to attend school.
Because of the help of MCF's training center, Felister has been able to save money and plans to start her own business.
Through their partnership with WorldCrafts, artisans at the training center earn a fair wage for creating beautiful handcrafted purses and jewelry. The crafter's dignity is being restored as they learn their own great worth.
Deaf people face incredible obstacles to finding work and sustainable livelihoods in Kenya and other developing nations. As part of the larger DOOR ministry (Deaf Opportunity OutReach), Sasa Designs empowers deaf women. These women struggle daily to overcome their physical limitations and provide for their families. Having been trained in beading, knotting, and wire-working techniques, artisans like Nancy and Peninah are now able to provide their families with plenty of nutritious foods, a consistent roof over their heads and education for their children.
Sema of Sema Leatherworks means "to speak" or "to say" in Swahili. The men artisans, who live in Nairobi, Kenya, work four days of the week on the products. They spend the remainder of the week applying their income to improve life for the people. They provide physical needs and offer encouragement and messages of eternal hope.
The cultural and artistic traditions of Central Asia have been preserved through the generations as women work together with their mothers and grandmothers creating masterful crafts. These items are as varied as the women who make them, with products reflecting the unique styles of individual crafters. Master's Handicrafts and WorldCrafts are honored to offer these fair-trade treasures to you on behalf of the indigenous artisans in the cities of Kyrgyzstan.
CraftAid, founded in 1982, provides jobs for people with disabilities (about 46 percent of current employees have disabilities.) Many of the artisans are hearing-impaired. CraftAid also employs many women responsible for providing for their families. CraftAid changes lives. Raima, a hearing-impaired artisan for CraftAid, found friends and contentment through her work. She also found Tana, her husband. Now they are married and have a son. Through their work, they can provide for their young family in Mauritius.
A native of Myanmar's infamous mining town began Mogok Inklings. Like most children, she played in the dirt, but unlike most, among the rocks and pebbles in her yard, the earth yielded fragments of rubies and semiprecious stones. Ironically, Mogok's underground riches, do not afford hopeful livelihoods or opportunities for its inhabitants. Consequently, the town's young population has been drawn away by the empty, better-life promises beckoning from the Thai and Chinese borders. Fueled by faith that has grown since her childhood, the now grown-up little girl acted on the idea of creating homegrown jobs using the smaller gemstones cast aside in the greed of the mining industry. Mogok Inklings now employs young people striving to finish school, and seeks to share hope with the artisans.
Blessed Hope provides creative work, skills development training, and true lasting hope to the women artisans and their families throughout the Himalayan Mountains of Nepal.
Labor and sex trafficking are both prevalent within Nepal, and the jobs and training Blessed Hope provides help eliminate the desperate situations which often lead to susceptibility to trafficking.
Through their partnership with WorldCrafts, the women artisans are paid salaries that make them able to purchase basic necessities for their families such as their children's school fees, diapers, electricity, and propane gas to cook with and heat their homes.
In the mountains of North Africa, the Berber artisans of the Leather Project skillfully craft leather scraps from a furniture and upholstery company into smooth bags. This time-honored tradition helps them to earn money that supplements their jobs as subsistence farmers and shepherds. More and more, Berbers are forced to move to the city to look for business jobs, but this artisan group allows them to stay with their families in the mountains and provide for their needs.
As markets open up, the women of Igira Impuhwe [i-GI-ru im-HU-we] in Rwanda earn needed income from the sales of their eco-friendly, handwoven grass baskets. Igira Impuhwe lifts women like Mukangenzi Laurance out of desperate poverty. She now rents a home, has health insurance for her family, bought clothes for her children, and rents a garden to grow food. Each woman in the group is also presented with the life-changing offer of eternal hope. Mukangenzi thanks God for changing her life. Igira Impuhwe means "God is compassionate" in Kinyarwanda.
More Than Sparrows artisans in Kigali, Rwanda, are growing in strength of spirit as well as new-found respect and dignity because they have worked together to leave their old lives behind. Discovering that they can learn new handcrafts which are valued by others has offered the artisans hope and the opportunity to prosper.
In 2013, a benefactor shared a reading from Matthew 10:31 with the artisans: "Fear not, therefore, you are of more value than many sparrows." These women were sex workers and street vendors, isolated women who were considered the lowest of the low, but this reading assured them that they had great value. Knowing their own worth brought them comfort and hope. Through their struggles they have discovered a sense of identity.
The artisans support each other, look after each other, listen to each other and take pride in what they do. They have become resilient and because of that, they now stand up and say, "We are More than Sparrows."
Living in fear of their ancestors, the Zulu people of KwaZulu-Natal, South Africa, continually slaughter cows, goats, and chickens as sacrifices. They even attribute any illness, death, or calamity as a mood of an ancestor's spirit that requires a sacrifice to appease. But, the people involved in the artisan group Wezandla, meaning "hands" in Zulu, have the opportunity to break free from this slavery. Each item they bead secures a paycheck for their poor families and the hope of freedom from fear.
Hill Top Crafts in Chiang Rai, Thailand employs Hmong women who sew unique, handmade purses. The artisans employed at Hill Top Crafts send their much-needed income home to their families in small villages in Laos and Vietnam. They are provided with shelter, as well as leadership training and discipleship courses. Their work at Hill Top Crafts helps them build self-esteem, encourage self-confidence, and provides essential hands-on skills to be successful in life. The women donate a portion of their sales to provide food, clothing, and shelter for orphans.
For years, Thai Country Trim in Thailand has been a safe haven for battered women to receive emotional and financial support.
One woman was constantly abused by her family until she sought help at Thai Country Trim. Her family gave her the most chores around the house and consistently told her she was worthless. Now, this woman, along with about 300 other women, is employed by Thai Country Trim, handcrafting items to earn a living and learn to heal from her past.
The talent and tranquility of The Well artisans belie their astoundingly difficult backgrounds: one was sold into prostitution by her family as a small child; several were teenage bar workers; and another was a single mom with no resources. They have discovered their own innate talents through careful training in Thai arts, and The Well has given them a sense of hope and a positive vision for their futures. Today, these women have dreams of opening their own businesses; becoming doctors, nurses, lawyers, and teachers; and sending their children to school.
Anadoule artisan group is located in the Turkish central city of Anatolia. It provides opportunities for artisans to learn skills in handmade crafts in order to provide income for their families and to keep the Turkish culture alive.
Each artisan has a unique story. Faitma is an artisan who needed an income to provide for her family. Her husband no longer worked, and she had several children. The work of handcrafting these products provides the earnings Faitma desperately needs for her family. Anadoule makes a lasting impact on the lives of these Turkish artisans and their families.
Widowed. Destitute. HIV-positive. These words could be used to describe the women of the Proverbs 31 Women artisan group in Uganda. But as they make colorful, attractive beaded jewelry out of recycled paper—you'd never guess—their hearts join together in song and their faces light up with smiles of joy! Sales of their beautiful, handmade items provide badly needed income to meet their families' most basic needs, such as food and rent. Today, words that best describe these Proverbs 31 women would be hopeful, encouraged, full of thanksgiving, and praise!
Baptist Friendship House in New Orleans, Louisiana, works to meet the physical, emotional, and spiritual needs of the homeless, human trafficking survivors, and those living in poverty.
They are a registered Christian Women's Job Corps site of national WMU.
The artisans learned to create pottery as a part of the therapy provided at Baptist Friendship House. Now these women have turned their hobby into a skill they can use to rebuild their lives.
Leaving their homes and coming to a new land with new customs, new rules, and a new language is overwhelming for many refugees. The language barrier creates obstacles as they try to begin a new life. Begin Anew, a Christian Women's Job Corps ministry in Nashville, TN has a mission "to empower individuals in Middle Tennessee to overcome obstacles caused by poverty by providing education, mentoring, and resources."
Begin Anew's south Nashville site works with refugees to teach them English and to help them become established in their new home. As a WorldCrafts artisan group, Begin Anew teaches a group of women how to hand screen-print a variety of items to earn additional income for their families.
In the words of Thee, one of the artisans from Myanmar, when you purchase an item handcrafted by Begin Anew, the artisans send you, "Big, Big Thanks!"
Kristina came from a background of abuse and drugs. After crying out to God to save her in her early 20s, she started a new chapter in her life by joining the Christian Women's Job Corps (CWJC) of Rusk County, Texas. Tutors helped Kristina complete her GED, allowing her to pursue her dream of becoming a nurse. CWJC of Rusk County exists to give women like Kristina a chance to get back on their feet. Participants can receive instruction in basic job skills, GED and literacy tutoring, and English as a second language courses. They also have the opportunity to earn supplemental income by working in the small jewelry and crafts business at the site.
Graffiti 2 Works coaches adults to develop the physical, mental, emotional, social, and spiritual skills necessary to achieve their best. Learning to sew and sell products gives these artisans jobs and builds their self-esteem.
As the artisans of Graffiti 2 Works sew together, they develop relationships with each other and with our Father.
Fleeing persecution in their native countries, such as Sudan, Bhutan, Burma, and Egypt, refugees often encounter new and threatening challenges upon arrival in the United States. Children are drawn into crime, parents are treated with disdain and overworked, and even basic tasks seem like impossible puzzles. Refugee Beads exists to provide hope, a sense of community, and needed income for refugees living in the Atlanta area. The jewelry created by these women is marketed across America to benefit their families. Many of the women are the wives of refugee pastors serving their own people groups.
Refugee Sewing Society in Clarkston, Georgia, teaches viable skills to vulnerable refugee women. The women receive payment for the products they sell, helping them earn a supplemental income. They have access to available resources including ESL and citizenship classes, which will allow them to adapt to their new home in America.
Sex trafficking is the fastest growing criminal industry. Trafficked women feed a worldwide demand for sex slaves, prostitution, and pornography. Poverty and the fear of uncertain futures leave these women vulnerable to the lure of traffickers. In the midst of this criminal world,
The WellHouse fights human trafficking as it extends our Father's grace and creates opportunities for restoration to exploited women. Here, trafficked victims enter a safe residential environment where they receive spiritual, mental, emotional and physical support. By purchasing a bracelet or necklace created by a human trafficking survivor, you join the battle against exploitation and share hope, love, and courage.
As West Bank artisans shape olive wood, they know each unique item means more than money earned. Carved by their hands, each product is inspired. And for these UEMS artisans, creating their art is not just a matter of shaping a piece of olive wood; it's a powerful testimony of hope delivered from one generation to another for more than 2,000 years.


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> Rago Calendar
Out and About 2019
The Coins of Maundy Thursday
Rago and Wright are Joining Forces
Fit for an Emperor
Modern Glass
Post-War/Cont. Art
19th/20th C. Art
Fine Furn./Dec. Arts
Historical/Militaria
Jewelry/Silver
Joshua P. Smith: A Personal Collection of 20th Century Art
by David B. Smith: Artist and son of Joshua P. Smith
I am pleased to present this selection of Contemporary 20th Century prints, photographs, paintings, drawings and sculpture acquired between 1975 and 2005, primarily from artists' galleries, by my father Joshua P. Smith. Joshua was an independent collector who curated highly praised shows such as The 1980s: Prints from the Collection of Joshua P. Smith at the National Gallery of Art, The Photography of Invention: American Pictures of the 1980s at the Smithsonian Museum of American Art, and exhibitions at the Katonah Museum of Art and the Corcoran Gallery of Art. His rigorous research, intuitive eye and deep connections ensured the works he acquired continue to inspire viewers in prestigious museum, university, and corporate and private collections around the world.
My sister Rebecca and I (pictured behind our father in the image above) grew up surrounded by hundreds of classic, eclectic and avant-garde artworks. Hung salon style, these works played off each other in unexpected ways, suggesting conversations among artists working at different times and in different media - conversations Joshua worked to foster and preserve. Fascinated by artistic minds and practices, I majored in Art History at Oberlin College, earned an MFA from Bard College, and became an artist myself. A solo show of my fabric-based works is currently on view at Halsey McKay Gallery in East Hampton, NY, with another upcoming in March 2019 at Geary Contemporary, New York, NY.
My father did not identify as an artist. Rather, he expressed himself through deep consideration, appreciation and understanding of artistic works. His relationship with the art he loved was intimate, as demonstrated by his ability to dutifully organize and highlight threads of meaning and style through movements and over time. He was at his best hanging out with artists in the East Village and hanging shows at the National Gallery of Art.
Below, I will highlight a few works from my father's collection on offer at Rago Auctions on November 10, with a particular focus on their importance to me, my father, and to their contribution to the conversation within the collection.
Lot 768 – Sigmar Polke
Estimate: $65,000 – 85,000
Sigmar Polke was Joshua's favorite artist and this piece was the most significant he acquired - another in the edition was featured in the recent retrospective at the Museum of Modern Art in New York. Joshua was drawn to the combination of formal experimentation and humor used to confront issues such as trauma, loss and confusion. In this case one potato can be seen as searching for, yet never crossing paths with, another.
Lot 752 – York der Knoefel
Estimate: $600 - 800
One area of specialty for Joshua was Post-War West and East German art. He was drawn to struggle and intense energy. Der Knofel's works are among the most extreme examples of raw emotion in the collection, but it is common to many works and starkly present in the German Neo-Expressionist works by Antonious Hockelman, Gunter Damisch, A.R. Penck, Franz Hitzler, Arnulf Rainer and Dieter Roth, among others.
Lot 812 – Andy Goldsworthy
Estimate: $5,000 – 7,000
Josh loved works that blurred lines, confused you at first glance and had a strong, inventive and almost performative sensibility. Andy Goldsworthy's works are an example of such. To create this series, the artist would lie down beneath a rainstorm to create an ephemeral document of his body in time and space. The photographs that he took after the storms passed are all that remains; a memory of this fleeting and deceptively powerful moment.
Lot 761 – Nikki Lee
Estimate: $800 – 1,200
Nikki Lee also used performance to make her photographic projects. By portraying herself in various social groups she speaks to photography's ability to blur the line between truth and fiction and explores issues of subcultures and identity. My father was often inspired by such conceptual projects; a thread of conceptualism runs through the work of many of his favorite artists, including Robert Heinecken, Kurt Peter Buchwald, Gunther Forg, Hiroshi Sugimoto and Robert Smithson.
Lot 711 – William Klein
The people in William Klein's iconic documentary photos burst with life and character. His subjects, whether in New York, Paris, Tokyo, Rome and Moscow, often seem to be knowingly performing and controlling their own images. His style translates perfectly to the realm of fashion photography, where he is widely seen as an innovator and influencer.
Lot 811 – Lewis Baltz
Lewis Baltz perfected a cool, detached and quiet voice that was central to the New Topographic movement. Far from the streets of the city, Baltz trained his large format camera on new suburban developments, often during construction or before they were occupied. His is a formally stunning criticism into the suburbanization of the American West.
Lot 790 – Richard Hambleton
In the 80s, Joshua spent considerable time among the artists and gallerists in the East Village, including Richard Hambleton, who was, at the time, primarily painting dark figures in doorways and on walls. He was an artist that moved away from the spotlight, but whose works have emerged as arresting and powerful creations of the era. Other works in the auction acquired by my father during this time are those by Brian Wood, David Sandlin, Amy Sillman, and Jean Holabird, Tony Towle, and Joseph Nevechtal among others.
Lot 806 – Fred Sandback
Estimate: $1,200 - 1,800
Sandback's work, while seeming quiet at first, contains a crackling energy within the taut and rich lines, making these works some of the most powerful in the collection. Other related works in the collection include those by Bjorn Copeland, Peter Downsbrough, Xylor Jane and Gunter Tuzina.
Lot 745 – Maureen Gallace
Estimate: $8,000 – 12,000
Gallace's work is painted with grace and simplicity, yet there is something slightly unsettling under the surface. It's this mysterious feeling that keeps me coming back to these works - wondering what lies behind the seemingly comfortable facades.
See these, and dozens of other works from the collection of Joshua P. Smith, in the online catalogs for our November 10 Fine Art Auctions, here.

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Aleksandr Naoemovitsj Froemkin (Russisch: ) (Chișinău,  – Toela, 27 mei 1976) was een Russisch scheikundige op het gebied van de elektrochemie.

Biografie
Froemkin werd geboren in Chișinău in het toenmalige Russische keizerrijk (tegenwoordig gelegen in Moldavië). Zijn vader was een verzekeringsagent. Later verhuisde het gezin naar Odessa, waar hij het gymnasium bezocht. Hij studeerde vanaf 1912 scheikunde in Straatsburg en Bern bij Volkmar Kohlschütter, en vanaf 1915 bij Aleksandr Sachanov aan de Universiteit van Odessa. In 1919 behaalde hij er zijn habilitatie.

Sinds 1916 was hij werkzaam in een laboratorium van een staalfabriek in Odessa, waar zijn interesse in de elektrochemie ontstond. Van 1920 tot 1922 was hij hoogleraar aan het Instituut van Volksopleiding in Odessa. Froemkin verhuisde in 1922 naar Moskou waar hij de leiding kreeg over de afdeling oppervlaktes van het Karpov-instituut voor Fysische chemie.

In 1930 werd hij benoemd tot hoogleraar elektrochemie aan de Staatsuniversiteit van Moskou, waar hij in 1933 de faculteit elektrochemie oprichtte en hij tot zijn overlijden in 1976 leiding aan gaf.

Froemkin trouwde driemaal, onder meer met Vera Inber.

Werk
Zijn eerste onderzoek publiceerde Froemkin reeds in 1914, en vanaf 1919 voerde hij onderzoek uit naar elektro-capillaire reacties aan elektrodes. Zijn belangrijkste bijdrage betrof de fundamentele theorie van elektro-reacties, die de invloed beschrijft van de structuur van de interface tussen elektrode en oplossing op de snelheid van de elektronenoverdracht. Hierbij introduceerde hij het begrip van nulladingspotentiaal, een van de belangrijkste eigenschappen van een metaaloppervlak.

Russisch scheikundige

redpajama_set_name: RedPajamaWikipedia


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10.3390/nano4030827
Lo, V. C.
Ren, Q.
Pham, C. L. L.
Morris, V. K.
Kwan, A. H.
Sunde, M.
Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability
Victor C. Lo
Qin Ren
Chi L. L. Pham
Vanessa K. Morris
Ann H. Kwan
Margaret Sunde
Discipline of Pharmacology, School of Medical Sciences, The University of Sydney, Sydney NSW 2006, Australia
School of Molecular Bioscience, The University of Sydney, Sydney NSW 2006, Australia
Nanomaterials 2014, 4(3), 827-843; https://doi.org/10.3390/nano4030827
Received: 23 June 2014 / Revised: 22 August 2014 / Accepted: 5 September 2014 / Published: 17 September 2014
(This article belongs to the Special Issue Self-Assembled Nanomaterials)
Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS∆15 and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS∆15 layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.
hydrophobin; protein; film; amphipathic; self-assembly
Hydrophobins are small, surface active proteins that are secreted from filamentous fungi and which self-assemble spontaneously into amphipathic layers at hydrophilic-hydrophobic interfaces [1,2]. These protein assemblies facilitate and support the fungal lifecycle at air-water and water-environmental interfaces. Hydrophobins are characterized by the presence of eight cysteine residues and the family members broadly fall into one of two main groups, the Class I and the Class II hydrophobins. Class I hydrophobins self-assemble into amphipathic layers that are composed of laterally associated and chemically robust fibrillar structures known as rodlets [3,4,5,6,7]. Class II hydrophobins self-assemble into non-fibrillar films that can be dissociated by treatment with alcohol solutions and detergents [8]. These biocompatible, self-assembling, amphipathic films show promise for use in nanotechnology applications, including coating implanted medical devices, biosensors and cell growth surfaces [9,10,11,12,13]. An understanding of the molecular basis for hydrophobin self-assembly is required in order to control the morphology and properties of these protein layers. The soluble forms of the hydrophobins share a similar structural scaffold imposed by the network of disulfide bonds, while sequence diversity between family and class members is accommodated in diverse secondary structural elements and loop regions [14]. The hydrophobins have relatively large, exposed hydrophobic patches on the protein surface and display a clustering of surface charged residues that is likely to underpin the observed high surface activity of these proteins [15,16]. We have carried out a study of the structure and stability of the layers formed by spontaneous assembly of Class I and Class II hydrophobins and an engineered chimera, which carries the rodlet-forming region of a Class I protein grafted onto the core structure of a Class II hydrophobin. The Class I hydrophobins we examined are DewA, a hydrophobin expressed in the spores of Aspergillus nidulans, and EAS∆15, an engineered variant of EAS (named for its easily wettable spore phenotype), the hydrophobin that forms a protein coating at the surface of the spores of Neurospora crassa. EAS∆15 lacks 15 residues from the long disordered region of EAS but forms rodlets with indistinguishable morphology and structural characteristics [5,17,18]. In spite of significant differences in sequence, size and molecular structure of the soluble forms of these Class I hydrophobins, DewA and EAS∆15 form rodlet films with very similar morphology. We have also examined self-assembly by a Class II hydrophobin from Neurospora crassa, NC2 [19] and an engineered chimeric hydrophobin NChi2, which carries the rodlet-forming region of EAS (this region is common to EAS and EAS∆15) grafted onto the core structure of NC2 [20]. Comparison of the protein layers formed by EAS∆15, the Class II protein NC2 and the chimeric protein NChi2 on highly oriented pyrolytic graphite (HOPG) shows that there are distinct differences in morphology and chemical resistance. While EAS∆15 rodlets are stable towards ethanol, 3M NaOH and 3M HCl washes and require treatment with trifluoroacetic acid (TFA) for dissociation, NC2 and NChi2 protein layers form a mesh on the HOPG surface, which is rapidly dissociated by treatment with ethanol but shows some stability towards 3M NaOH and 3M HCl. These characteristics will facilitate the use of these self-assembled amphipathic nanomaterials for biotechnological purposes.
2.1. EAS∆15 and DewA Class I Hydrophobins Form Rodlets with Similar Morphology in Spite of Differences in Protein Sequence and Solution Structure
We have previously shown that the solution monomeric structures of EAS∆15 [17] and DewA [5] display quite different secondary structure and loop elements (Figure 1a,b). Here, we have investigated the role of the hydrophilic: Hydrophobic interface in self-assembly by these two hydrophobins and compared the morphology of the protein layers formed by EAS∆15 and DewA. The rodlets formed by Class I hydrophobins have an amyloid substructure and therefore rodlet formation can be monitored through the increase in fluorescence that arises from binding of the amyloid-specific dye Thioflavin T (ThT) to the rodlets as they form [18,21]. We have used a ThT fluorescence-based in vitro system to monitor rodlet assembly by EAS∆15 and DewA. When the proteins are incubated with agitation in the absence of an air-solution interface (by completely filling the wells so that the plate sealing film is in contact with the surface of the solution), no ThT-positive rodlet assembly is detected. When an air gap (i.e. an air-solution interface) is introduced in the wells by reducing the volume of this protein solution, continued incubation with agitation results in rapid rodlet assembly (Figure 1c). This demonstrates that rodlet assembly by the Class I hydrophobins EAS∆15 and DewA only occurs at a hydrophilic-hydrophobic interface. These results contrast with those of Zykwinska et al. [4] and Longobardi and colleagues [22], who have demonstrated that the hydrophobins SC3 and Vmh2 can assemble into rodlets in solution. Transmission electron microscopy (TEM) images of EAS∆15 and DewA rodlet layers transferred from the surface of protein droplets onto holey films, so that the protein layers can be imaged in the absence of a supporting film, show that these two proteins form rodlets with similar dimensions and substructure (Figure 1d,e). The rodlets have a "double track" appearance; EAS∆15 rodlets are 6.7 ± 1.3 nm wide and DewA rodlets are 6.7 ± 0.8 nm wide, which is consistent with the model for intermolecular assembly that has been proposed for EAS rodlets [20]. When a solution of DewA is dried down onto a HOPG surface, the protein spontaneously self-assembles into rodlets (Figure 1f) with similar morphology to that seen for EAS∆15 [13] and other Class I hydrophobins [3,4]. Extensive structural studies are yet to reveal whether or how DewA is accommodated within a similar rodlet structure [5] but these TEM and atomic force microscopy (AFM) studies suggest that similarities in the assembly mechanism are likely.
We have previously demonstrated that surface tension controls the spontaneous self-assembly of hydrophobins at the air-solution interface [23]. This work demonstrated that the addition of additives that reduce solution surface tension results in an increase in the time taken for the Class I hydrophobin rodlets to self-assemble; and in some cases, rodlet assembly was completely inhibited over the timescale of the experiment. From these results it was apparent that the Class I hydrophobins EAS∆15 and DewA show distinct sensitivity to the solution surface tension, indicative of the individual propensity of each of these surface-active proteins to migrate to the interface and undergo the conformation changes that result in self-assembly into the fibrillar form. We have used this sensitivity to surface tension to optimize conditions for preparation of highly ordered fibrillar films from DewA and EAS∆15. Inclusion of 15% ethanol in the hydrophobin solution results in the formation of more ordered single layers (Figure 2a,b) while increasing ethanol concentration to 25% tends to result in the observation of many areas displaying a cross-hatched morphology (Figure 2c,d). In TEM, this observation is consistent with two single rodlet layers lying on top of each other. There are at least two possible reasons for this morphology. The increasing ethanol in solution may alter the way in which the excess protein solution is blotted from the grid, resulting in the formation of an additional rodlet layer as the grid dries. Or, the presence of ethanol may affect the adhesion of the rodlet layer to the grid, such that as the excess protein solution is removed by blotting, patches of rodlets are mobilised and slide across on top of each other.
Figure 1. Solution structures of the soluble forms of Class I hydrophobins: (a) EAS∆15 and (b) DewA, determined by nuclear magnetic resonance (NMR) spectroscopy. Ribbon representations of protein molecular structure were prepared from Protein Data Bank (PDB) entries 2K6A and 2LSH using the molecular graphics program PyMol [24]. (c) Rodlet assembly by EAS∆15 and DewA assayed by Thioflavin T (ThT) fluorescence demonstrates that these Class I hydrophobins only assemble into amyloid-like rodlets after introduction of a hydrophilic-hydrophobic interface. Transmission electron microscopy (TEM) of negatively stained: (d) EAS∆15 and (e) DewA hydrophobin rodlet monolayers, transferred from the surface of protein droplets and imaged through the holes in a holey film. (f) Atomic force microscopy (AFM) of the rodlets formed when a 5 µg/mL DewA solution was allowed to dry overnight onto a highly oriented pyrolytic graphite (HOPG) surface.
Figure 2. TEM image of negatively stained DewA and EAS∆15 hydrophobin rodlet-containing layers transferred from the surface of droplets of solutions containing: (a) DewA 15% (v/v) ethanol; (b) EAS∆15 15% (v/v) ethanol; (c) DewA 25% (v/v) ethanol; and (d) EAS∆15 25% (v/v) ethanol after incubation at room temperature for 20 min.
2.2. EASΔ15 Forms a Single Layer Composed of Rodlets on Highly Oriented Pyrolytic Graphite
The rodlets formed by EASΔ15 at an air-solution interface can be transferred to another hydrophobic surface, such as HOPG. Initially, EASΔ15-coated surfaces were prepared for AFM imaging using a modified protocol based on the sample preparation protocol for TEM. A droplet of EASΔ15 protein solution at a concentration of 25 µg/mL was incubated on PARAFILM® (Bemis Company Inc. Neenah, WI, USA) for 1 min at room temperature, allowing the protein to assemble into a rodlet coating on the surface of the droplet. Assembled rodlets were transferred onto a freshly cleaved HOPG surface by contact between droplet and HOPG surface for 5 min. The surface was then briefly washed with water and air-dried before imaging. Clusters of laterally associated rodlets ("rafts") were observed, dispersed across the HOPG surface (Figure 3a). The morphology of the rodlets observed under AFM was similar to that observed by TEM when hydrophobin layers from a droplet surface were transferred to carbon-pioloform-coated copper grids. The rodlets are hundreds of nanometres long, are distinct from the HOPG background, and are clearly visualized in both amplitude and phase images.
Class I hydrophobin rodlet formation occurs spontaneously at hydrophilic-hydrophobic interfaces, so it is also possible to assemble rodlets from solution directly onto the HOPG surface. Methods were developed which resulted in the formation of highly regular and uniform layers of EASΔ15 on HOPG. The most uniform and reproducible surface was obtained by drying a dilute protein solution by evaporation directly onto the HOPG surface at room temperature, over ~16 h, followed by extensive washing with Milli-Q® water (MQW, Merck Millipore, Bayswater, Australia). Initial imaging showed that when the EASΔ15 coating was prepared by drying down a 50-µL drop of protein solution (at a concentration of 5 µg/mL) onto the HOPG block, a thicker protein coating, indicative of the presence of multiple protein layers, was formed on the surface (Figure 3b). The upper layers could be progressively removed by extended periods of washing, as can be seen in Figure 3c,d. When the surface was rinsed with MQW for 1 min, the upper layer(s) were disturbed and partially removed, resulting in the observation of rafts of rodlets scattered on top of the rodlet layer across the entire image (Figure 3c). Finally, when the surface was washed for 7 min with a stream of running MQW, a very uniform single layer of protein rodlets could be obtained (Figure 3d). Almost all of the examined surface area is covered with rodlets, which are microns long, unbranched and all oriented in one direction. The rodlets formed under these conditions by EAS∆15 are longer and display a more regular lateral packing than those reported in the literature from other Class I hydrophobins but have similar width and height [4,6,25].
Figure 3. (a) AFM topographic scan of HOPG surface coated with a layer of EAS∆15 protein after a 50-µL drop of EAS∆15 (25 µg/mL) was incubated for 1 min and transferred onto a freshly cleaved HOPG surface; multiple protein rafts are observed; (b) AFM scan of HOPG surface coated with a layer of EAS∆15 protein after a 50 µL drop of EAS∆15 (5 µg/mL) was left to dry overnight onto a freshly cleaved HOPG surface. Sample was imaged directly after drying; (c) a 1-min wash to remove loosely bounded protein layers reveals an underlying, ordered single layer rodlet film; and (d) after 7 min of washing with a stream of running Milli-Q® water (MQW), loose fibril layers are removed and a highly ordered layer of rodlets remains attached to the HOPG.
Stability of the EASΔ15 Rodlet Layer
There have been many reports that indicate Class I hydrophobin rodlets are extremely robust towards chemical treatments and can only be dissociated to a monomeric form by treatment with concentrated TFA or formic acid [20,26]. Indeed, this has been taken as one of the defining features of Class I hydrophobins, since it contrasts with the films formed by Class II hydrophobins, which can be disrupted by treatment with alcohol or detergent solutions [26]. However, most of these characterizations have been made on the basis of treatment of pre-assembled hydrophobin rodlets in solution with a range of chemicals followed by analysis of the solution using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Hydrophobin rodlets are not dissociated or solubilized by treatment with reducing sample buffer and therefore do not electrophorese through a SDS-PAGE gel. However, the monomeric forms of the hydrophobins can be mobilized and visualized by SDS-PAGE [27,28]. In order to directly examine the chemical stability of the rodlet layers, AFM was used to visualize the morphology of the rodlets upon treatment with 60% or 100% (v/v) ethanol, 3M NaOH, 3M HCl, and 100% TFA. These test conditions were chosen for investigation as they have been used to probe the stability of hydrophobin assemblies in other studies [29,30]. The EASΔ15 coatings were prepared as described above, by drying a dilute protein solution onto HOPG, followed by washing for 6 min with running MQW. The surfaces were then submerged in the test solution for 5 min before further washing with MQW to remove any loosely bound protein.
Treating the EASΔ15 rodlet films on HOPG with 60% or 100% (v/v) ethanol efficiently washed off any loosely bound protein but did not disturb the layer in direct contact with HOPG. The process resulted in increased uniformity of height across the HOPG-bound rodlet layer (Figure 4a). Treatment with 100% TFA completely disrupted the fibrillar morphology of the rodlet layer and reduced adherence of the protein to the graphite surface (Figure 4b). The loss of the fibrillar structure was accompanied by the aggregation of the protein into large, globular structures. Treatment of the rodlet layer with 3M NaOH or 3M HCl did not generally dissociate the rodlet layer from the HOPG or disturb the fibrillar structure as seen by the presence of the underlying fibrillar structure in the images (Figure 4c,d). However, residual salts or impurities remained attached to the layers that were not readily removed by washing with water.
Figure 4. AFM scans of EAS∆15 rodlets on HOPG treated for 5 min with different solvents, followed by a 2-min water wash: (a) exposure to up to 100% ethanol does not affect the structure of the EAS∆15 rodlet layer and effectively cleans the surface of any loosely bound protein; (b) EAS∆15 fibrillar film is disrupted by treatment with 100% trifluoroacetic acid (TFA); fibrillar rodlet structure is stable towards treatment with: (c) 3M NaOH and (d) 3M HCl, but residual impurities remain attached to the fibrils and are not easily removed by washing.
2.3. NC2 Self-Assembles into a Protein Mesh
We have previously reported the first study of the structure and self-assembly of the Class II hydrophobin NC2 from Neurospora crassa (Figure 5a; [19]). This protein shows a high level of sequence homology to Class II hydrophobins from other fungi and has the pattern of cysteine distribution along the polypeptide chain that is characteristic of other Class II hydrophobins. The protein has been shown to be highly surface active, consistent with a large hydrophobic area on one face of the protein and a clustering of charged residues on the opposite face of the protein [19]. When NC2 is prepared on HOPG under the same conditions as described above for EASΔ15, the protein forms an interconnected mesh with pores of diameter 20–30 nm and a height of 1.5–2 nm (Figure 5b). This morphology resembles that formed by the Class II hydrophobin HFBI when the films are prepared by compression with a Langmuir trough, which compresses the hydrophobin film laterally [8]. This results in a regular and uniform protein lattice with repeating hexagonal units of diameter 20–30 nm2.
Figure 5. (a) Ribbon representation of the solution structure of NC2, prepared from PDB Entry 4AOG using PyMol [24]; (b) NC2 layer washed with MQW for 5 min, displaying a protein network with pores of 20–30 nm and a layer height of 1.5–2 nm; (c) NC2 layer after treatment with 60% ethanol for 5 min; (d) NC2 layer after treatment with 3 M NaOH; and (e) NC2 layer after treatment with 3 M HCl.
Stability of the NC2 Layer
Other Class II hydrophobins have shown promise as biocompatible coatings in many biotechnology applications, including modifying electrodes for use in harsh chemical environments and as solubilising agents for poorly water-soluble drugs [11,31]. We have therefore characterized the structure and stability of the coatings formed by the Class II hydrophobin NC2, towards a range of solution conditions.
NC2 coatings were prepared as described in Section 2.2 and then incubated with 60% (v/v) ethanol, 3M NaOH, or 3M HCl at 25 °C for 5 min. After treatment, the surfaces were washed extensively with MQW to remove any solubilized and loosely bound protein, and then dried in air. As reported for other Class II hydrophobin protein coatings, the NC2 film was dissolved upon incubation with 60% (v/v) ethanol (Figure 5c). After treatment with ethanol, only small and scattered protein aggregates could be observed on the HOPG surface. The treatment with 3M NaOH and 3M HCl had a less drastic effect on NC2 assemblies, as more protein remained bound to the surface after treatment (Figure 5d,e). The morphology of the layers was disturbed by the extremely acidic and basic conditions, with larger uncoated HOPG regions visible after the treatments; however, the remaining protein layer displayed some level of organization and was not completely amorphous. While extremely acidic and basic conditions would be expected to affect salt bridges and hydrogen bonds, treatment with ethanol is likely to disrupt hydrophobic interactions. Therefore, these observations suggest that the NC2 monomers mainly interact with each other and form films via hydrophobic interactions. In addition, the NC2 layer binds to the hydrophobic HOPG surface via hydrophobic interactions, explaining why such a dramatic effect was observed upon ethanol treatment. By comparison, neither the lateral assembly within Class I rodlet films, nor the interface between protein layer and hydrophobic surface, is disrupted by ethanol treatment. This may be due to more extensive and stronger contacts between the constituent hydrophobins within the rodlets and also between the protein layer and the hydrophobic surface.
2.4. Self-Assembly by the Chimeric Hydrophobin NChi2 on Highly Oriented Pyrolytic Graphite
As part of our analysis of the sequence-structure relationships within the hydrophobin protein family, we prepared a chimeric hydrophobin with the main part of the protein composed of the NC2 protein and the sequence between the Cys7 and Cys8 replaced by the corresponding sequence from EAS (Figure 6a) [20]. This protein is able to self-assemble into short fibrillar structures, but only when incubated for extended periods at low pH and high temperature with shaking (Figure 6b), unlike EAS and EASΔ15, which readily assemble into rodlets under most native conditions. This work demonstrated that the region between Cys7 and Cys8 can drive protein self-assembly into rodlet-like structures when grafted onto the core structure of the Class II hydrophobin NC2. For practical reasons, it is not possible to replicate these solution conditions when preparing protein layers on HOPG. However, when the protein is dried down from a solution at pH = 2.5 and incubated at 45 °C, NChi2 self-assembles into a mesh-like protein layer that is more similar to the protein film produced by NC2 than to the rodlet layer produced by Class I hydrophobins (Figure 6c). The thickness of the layer is ~2 nm, which is consistent with the thickness of a single hydrophobin layer. However, in some areas of the same sample a protein layer of ~4 nm was observed, possibly a bilayer of NChi2 that is stable enough to resist extensive washing by water.
Figure 6. (a) Sequence alignment illustrating the construction of the chimeric hydrophobin NChi2 from the proteins NC2 and EAS (named for its easily wettable spore phenotype). The Cys7–Cys8 region of NC2 is shorter than the corresponding region in EAS and EAS∆15, this difference is indicated with a dotted line. The 15-residue deletion from EAS, to generate EAS∆15, is indicated by a solid line. The conservation of the amyloidogenic region between EAS, EAS∆15 and NChi2 is indicated in red; (b) TEM image of negatively stained NChi2 rodlets formed under conditions of low pH (=2.5) and elevated temperature (45 °C) with extended shaking. (c) AFM image of NChi2 dried down from pH = 2.5 at 45 °C and then washed with MQW for 5 min.
Stability of the NChi2 Layer
Drying a solution of NChi2 in water onto HOPG under native conditions resulted in the formation of layers with a Class II-like morphology (Figure 7a). Treating NChi2 films formed in this way with 60% (v/v) ethanol disrupted the morphology of the NChi2 coating to a certain extent, but did not fully solubilise the layer (Figure 7b). Treatment with 3M NaOH did not appear to significantly disturb the mesh morphology (Figure 7c). A much larger change in the morphology of the layer was observed when the sample was incubated with 3M HCl (Figure 7d). In some areas of the acid-incubated NChi2 layer, the protein network was significantly disturbed and became substantially thinner, with fewer inter-protein connections visible.
Production of rodlets by NChi2 requires extended periods of incubation at elevated temperatures, conditions that are difficult to achieve during preparation of the surface from a small volume protein droplet. It is therefore likely that the NChi2 layers formed on HOPG do not contain the intermolecular β-sheet structures that are found in the Class I rodlets, and which are likely to contribute to the stability of the rodlets.
Figure 7. Morphology and stability of NChi2 layers formed from protein at under native conditions: (a) NChi2 coating washed with MQW has Class II morphology; (b) treatment with 60% ethanol does not disrupt the layer; (c) the protein network attached to HOPG is stable towards 3M NaOH; and (d) treatment with 3M HCl disrupts interactions between protein molecules, loosening the mesh structure.
3. Experimental Section
3.1. Hydrophobin Production
All hydrophobins were produced recombinantly in E. coli BL21 (DE3) as His6-ubiquitin-hydrophobin fusion proteins, then refolded in vitro to a functional structure with four intact disulfides and cleaved from the His6-ubiquitin fusion tag before final purification by reverse-phase HPLC and confirmation of correct folding by 1H one-dimensional nuclear magnetic resonance (NMR) spectroscopy. Expression, purification and refolding was carried out as described for EASΔ15 [17] DewA [5], NC2 [19] and NChi2 [20]. Protein concentration was determined by Pierce BCA bicinchoninic acid assay (Thermo Fisher Scientific, Scoresby, Australia) or using the appropriate extinction coefficient where the protein contained aromatic residues. Samples for TEM and AFM were prepared in filtered MQW at the desired concentration.
3.2. Thioflavin T Assay
ThT assays were performed with 25 µg/mL hydrophobin and 40 µM ThT in 50 mM Tris. HCl, pH = 8.0 (for EAS∆15) or 50 mM sodium acetate, pH 5.0 (for DewA). To investigate the role of the interface, microplate wells were filled to overflowing with hydrophobin/ThT solution (>400 µL) and then wells were sealed with sealing film. The complete removal of air from the wells was achieved by adding additional solution into the well by injection with a needle through the film. The plate was then resealed. The assay was performed at room temperature in a BMG LABTECH POLARstar Omega Microplate Reader (Mornington, Australia) with 440–10 nm excitation filter and 480–10 nm filter for detection of emission. Double orbital mixing mode at 700 rpm was continued between every data collection point. After 200 min of incubation, 100 µL of the protein solution was removed by aspiration to introduce an air-solution interface and agitation was continued.
3.3. Transmission Electron Microscopy
For TEM a drop of hydrophobin solution (20 µL of 50 µg/mL of DewA or 20 µL of 100 µg/mL of EASΔ15) was incubated in air at room temperature for 20 min to allow self-assembly on the air-solution interface. A carbon and formvar-coated copper microscopy grid (200-mesh, ProSciTech, Townsville, Australia) or holey carbon grid (200-mesh, ProSciTech) was floated on top of the protein drop for 1 min, then excess solution was removed by touching the edge of the grid to filter paper. The grid was then washed by touching to three drops of MQW, with removal of water by wicking with filter paper between drops, and then stained with 2% uranyl acetate solution for 10 min. Excess stain was wicked off, and the grids were air dried. Grids were analyzed with a Philips CM 120 Biofilter TEM (Philips, Eindhoven, The Netherlands) operating at 120 kV and images were collected with the Gatan Imaging Filter System and Camera (Warrendale, PA, USA).
3.4. Atomic Force Microscopy
For AFM analysis of hydrophobin layers formed on the surface of droplets, a droplet of EASΔ15 protein solution at a concentration of 25 µg/mL was incubated on Parafilm® for 20 min at room temperature then transferred onto a freshly cleaved HOPG surface by contact between droplet and HOPG surface for 5 min. The surface was then briefly washed with MQW and air-dried before imaging. For preparation of protein layers in situ on HOPG, lyophilised hydrophobin protein was dissolved in filtered MQW to a final concentration of 5 µg/mL, and then a 50-µL droplet was placed on the surface of a block of freshly cleaved HOPG (Holgate Scientific, Terrigal, Australia), which was obtained by peeling off the top layer of the HOPG block by adhesion to sticky tape. The protein solution was incubated in air at room temperature with protection from dust, and allowed to dry completely. Coated surface was then washed with a stream of running MQW for 6 min (EASΔ15 and NChi2) and 2 min (NC2), and then air-dried. To prepare NChi2 samples at pH = 2.5 and 45 °C, lyophilized protein was dissolved in 50 mM glycine (pH = 2.5), and a 50-µL drop of the sample was dried on freshly cleaved HOPG surface at 45 °C, and the coated area was washed with MQW for 6 min, and then air-dried. Any variations in sample preparation are indicated in the legends of relevant figures.
The morphology of the prepared HOPG surface was characterized at ambient atmosphere, using a Multimode Nanoscope® III AFM (Veeco, Santa Barbara, CA, USA) operated in tapping mode. The AFM probes used were silicon-scanning probes with a tip radius <10 nm, operated with a force constant of 40 N/m and resonant frequency of 300 kHz (Tap300AI-G, BudgetSensors™, Innovative Solutions Bulgaria Ltd., Sofia, Bulgaria). Analysis of AFM images was performed with the Gwyddion Software (Czech Metrology Institute, Brno, Czech Republic [32]).
Despite a similar disulfide-linked structural scaffold, Class I, Class II and chimeric hydrophobins self-assemble at interfaces into single protein layers with distinct morphologies (Figure 8).
Figure 8. Schematic representation of the monomeric and self-assembled forms of Class I, Class II and chimeric hydrophobins. Grafting of the fibril-forming segment from a Class I hydrophobin onto a Class II hydrophobin can confer additional self-assembly character, as evidenced by the chimera NChi2 which is able to form both a Class II-like polygonal protein network or Class I-like fibrillar rodlet structures depending on assembly conditions.
We have demonstrated that recombinant preparations of two Class I hydrophobin proteins readily self-assemble at hydrophilic-hydrophobic interfaces into layers with a very similar fibrillar structure despite the large differences in protein sequence and solution structure. Solution conditions can be tuned to generate highly ordered and uniform protein films. These Class I films adhere tightly to hydrophobic surfaces such as graphite and are stable to alcohol washes and to 3 M NaOH and 3 M HCl washes but can be dissociated by treatment with concentrated TFA. In contrast, the Class II hydrophobin NC2 readily assembles onto a hydrophobic surface to form an extended protein network that lacks fibrillar morphology. This protein film is not stable when treated with 60% ethanol but displays some resistance towards acid or base washes. We have demonstrated that the chimeric hydrophobin NChi2 can form either a fibrillar rodlet structure or a polygonal protein network, depending on incubation conditions. The NChi2 layers deposited onto HOPG under native conditions appear partially resistant to ethanol and base washes but are more disturbed by acid wash. The morphology of the NChi2 assemblies formed under conditions of low pH and elevated temperature is more similar to the morphology of the structures formed by NC2 than to that of the rodlets formed by EASΔ15 protein. However, the NChi2 assemblies formed in solution at low pH and elevated temperature show enhanced fluorescence upon ThT binding, indicating the presence of an amyloid structure. Overall, therefore, the incorporation of the rodlet-forming region from EAS results in more robust intermolecular interactions between NChi2 molecules when the protein undergoes self-assembly. This investigation of Class I, Class II and chimeric hydrophobins demonstrates that the stability and fibrillar nature of the Class I hydrophobins resides in the amyloidogenic region. This is required to impart stability towards extreme pH on the assemblies. This work indicates that if the amyloidogenic region is maintained, novel functionalities can be included within hydrophobins while maintaining the ability to form robust protein coatings. In contrast, the Class II scaffold can be used to prepare protein films which are stable towards pH extremes but can be dissociated by alcohol treatment. We have previously established that the amphipathic nature of hydrophobin coatings can be used to reverse the wettability of hydrophobic surfaces and to increase the biocompatibility of nanostructures such as carbon nanotubes which may have applications in aqueous biological environments [13]. The fungal hydrophobin proteins therefore show promise as amphipathic coatings on nanomaterials, with molecular engineering being used to generate novel properties and functionalities.
The authors gratefully acknowledge the support of the Australian Research Council Discovery Project Grant (DP1093949), Australian National Health and Medical Research Council (NHMRC) Support in the form of R. D. Wright Career Development Fellowship for Margaret Sunde and a University of Sydney's Vice-Chancellor's Research Scholarship for Vanessa K. Morris. We thank Chiara Neto for access to AFM instruments and acknowledge the facilities, and the scientific and technical assistance, of the Australian Microanalysis Research Facility at the Electron Microscope Unit, The University of Sydney.
Victor C. Lo collected all of the electron microscopy images and assisted with characterization and analysis and with writing and correcting the article. Qin Ren developed methods for preparation of reproducible hydrophobin films on HOPG and collected all of the AFM images. Chi L. L. Pham and Vanessa K. Morris prepared recombinant protein samples and assisted with correcting of the manuscript. Ann H. Kwan and Margaret Sunde designed the project, participated in the analysis of the results and wrote and corrected the article.
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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
Lo, V.C.; Ren, Q.; Pham, C.L.L.; Morris, V.K.; Kwan, A.H.; Sunde, M. Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability. Nanomaterials 2014, 4, 827-843. https://doi.org/10.3390/nano4030827
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Cooking Eating Smart
What Foods Are Pork Meats?
Pork is a term used to describe domestic pig meat. Today, it is one of the most widely consumed meats in the world. Pig husbandry dates back to at least 5000 BCE. Read on to learn more about the different types of pork. Are Doritos and Cheetos made from pork?
What foods are made from pork?
Pork is a versatile meat that lends itself to many recipes. However, it's important to be cautious when preparing it. If you don't take the right precautions, it can cause food poisoning. Some diseases associated with improperly prepared pork include trichinellosis.
There are more than 40 products made from pork. In fact, pork bones provide glycerin, which is added to some toothpastes. Pig skins are used to make gelatin, which is used as a thickening agent. The fat from pork bones is also incorporated into many toothpastes.
Pork is widely available in most grocery stores. It's sold in various cuts, including bacon and ham. You can also find pork chops, shoulder chops, and ribs. You'll find ham and tenderloin in delis around the U.S. Pork is a versatile meat and can be prepared in a variety of ways, including frying and grilling.
What meats are considered pork?
Pork is classified as a red meat by the United States Department of Agriculture. However, pork may be pale in color before it is cooked. This is due to the higher amount of myoglobin in the meat. Pork contains more myoglobin than fish and poultry. While pork is considered a red meat, it is often referred to as white meat in the culinary world.
Pork chops are the most popular cut of meat, which is cut from the upper part of the pig's body from the hip to the shoulder area. Pork chops may be thin or thick, and they can come in a variety of shapes. While pork is traditionally classified as a white meat, some nutritional studies classify it as a red meat. Pork is also rich in phosphorus and selenium, two nutrients that are needed for healthy living.
Pork meat has a unique flavor. It has more flavor than chicken, and its meat texture is often chewy. Pork is the most popular meat in the Western world, and is the most popular meat in Central Europe. Lean pork cuts are usually lower in calories than chicken or beef. Pork loin chops are another option. They are similar to center-cut loin chops, but have a different bone structure. The closest alternative is sirloin steak, which is also very similar to pork chops.
Is Doritos made of pork?
There has been some controversy over whether Doritos are made of pork. Although the main ingredient in Doritos is pork fat, it's not the only part of the snack. The seasoning mix is also made with pork by-products. The ingredients in this seasoning mix include gelatin (derived from cow bones) and pork flavoring. This is what gives the chips their spicy and hot taste. As a result, they are not considered halal and are considered haram.
The answer to this question depends on the brand. Doritos are manufactured by Frito Lay, an American subsidiary of PepsiCo. The company does not use swine enzymes to make their products, but some international varieties do. In addition to Doritos, the company also makes hot Cheetos, a spicy snack. The perfect blend of cheese and spices makes hot Cheetos a popular choice for snacking.
As a vegan, you may be concerned about the pork content of Doritos. Luckily, you can find vegan Doritos. However, you need to read the ingredient list carefully. There are two types of vegan Doritos, wavy and crispy. There's also a vegan brand called Takis.
Do Cheetos contain pork?
While it is difficult to know for certain whether Cheetos contain pork, the company responsible for making them uses animal-derived ingredients, including pork fat. Pork is often used as a flavoring agent in processed foods, usually in conjunction with MSG. Cheetos are not made with pork fat, but they do contain iso-alpha-acids, a key flavor molecule that can be found in pork. Pork derivatives are used in the flavorings of many processed foods, including beer, soda, and cosmetics.
Cheetos also contain the smoky flavor that many consumers love. This savory smoky flavor is derived from pork. The savory smoky flavor of Cheetos comes from its taste and flavoring. In order to be vegan, you need to make sure that you avoid foods that contain pork, or at least, those that contain pork.
If you're worried about eating pork or dairy, you can opt to switch to vegan snacks. For example, Earth Balance has a line of vegan snacks that include cheetos cheese puffs. But many cheese-flavored snacks contain animal-derived enzymes, and are not kosher. In addition, some Doritos may contain pork enzymes.
Are hot dogs pork?
A hot dog is a meaty sausage that is usually half meat and half other stuff. It may be skinless or stuffed in a casing. It's also known as a frankfurter, wiener, or a vienna. Hot dogs are traditionally made with beef or veal, but now they can also be made with chicken or vegetarian proteins. Pork hot dogs tend to have less fat and are more filling than beef-based ones.
In the U.S., hot dogs are regulated by the Food and Drug Administration. To qualify for this designation, hot dogs must be smoked or cooked, and they cannot contain more than 40% fat or water. In addition, hot dogs may contain up to 3.5 percent nonmeat binders. These binders may include grains, breadcrumbs, or powdered milk products. Whether a hot dog is made from pork or beef is ultimately up to the consumer, but it should be made from real meat.
Hot dogs are traditionally made with beef, though some producers produce all-beef versions that contain no filler. While hot dogs are popular all-beef creations, some people still think of them as pork-only items. In any case, it is important to check the labels before purchasing any.
Is chicken a pork?
Despite the similarity of the names of chicken and pork, the two are actually classified differently in the food industry. While pork is considered a red meat because of its high myoglobin content, chickens are considered white meat. According to the American Meat Society, meat is "any animal flesh, organ, or byproduct that is consumed for food." Other classifications include fish, poultry, and fish byproducts.
Pork meat is darker in color, and it has distinct marbling. Marbling is the visible white wavy lines of intramuscular fat that are characteristic of pork. In addition, pork is also considered a processed meat, since it is preserved using chemical preservatives. Pork comes in many cuts and varieties. Primarily, pork cuts include shoulder, ham, leg, and loin. These cuts differ in texture, taste, and aroma.
Is turkey a pork?
Pork and turkey share several similarities in their flavor, but they are not identical. In fact, they are inversely related in terms of umami notes. Both meats are high in saturated fat and sodium, which are known to increase the risk of heart disease. Pork and turkey meat also have different nutritional values, with pork having higher protein and less fat.
Turkey is the larger of the two species, which requires a greater amount of seasoning and brine to properly cook. Both meats are high in animal protein, and both can be found fresh or frozen. Both are classified as white meat, which means that they have lower iron content than red meat.
In terms of health, pork meat is higher in zinc, selenium, choline, and phosphorus. Turkey meat contains a slightly higher fat content, but tends to be lower in sodium.
Do Skittles have pork in it?
If you're a vegetarian, or are concerned about pork consumption, you might want to look up the ingredient label on Skittles. While the sweet candies are not sourced from pork, they do contain gelatin, which is made from animal collagen. This ingredient is used to give foods their chewy texture. The Wrigley Company has since removed this ingredient from the candy. But you should know that they still contain pork-containing ingredients, such as carmine and white sugar.
The sugar in Skittles comes from two sources: sugar beets and sugarcane. Beet sugar is considered a healthful ingredient, and it's free of animal products and additives. Palm oil is a major contributor to deforestation and negatively impacts forests around the world. In addition, it contributes to climate change. Therefore, if you're a strict vegan, you shouldn't eat Skittles.
A third ingredient in Skittles is gelatin. Gelatin is a protein made from collagen, the protein found in skin, connective tissues, and bones. Pigs are the most common source of gelatin, which is a key ingredient in gummy candies. If you're a vegan, you may want to check the ingredients list before buying the candy.
How to Make a Roux For Beef Stew
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Contact Sherri on 09 372 9898.
The main reason we're all here.
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Roll – up Wednesday – play starts 2.00pm, register by 1.30pm.
Bar facilities available from 2.30 pm.
Business House Bowls starts 1st Nov. 6pm start. If you are not in a team come along anyway to watch or fill in for teams with missing members.
Darts League 6pm to 8pm. $10 entry. Text 021771132 if interested in playing. Arrive before 6pm.
The club hosts a number of events throughout the year, some of which are open to the general public for example "Business House Bowls" and "Sponsored Fun Tournaments"
For those wanting a fun activity the club is available for social bowls, corporate groups, team building, hen and stag parties, birthdays, girls or boys weekends away or those wishing to just "have a go". Catering is available on request, equipment and tuition provided so no experience required and fun is guaranteed.
If weather not suitable for bowling, it's indoors for a fun game of pool and darts.
Bookings for these groups is essential, so contact the office manager on 09 372 7108, to discuss availability and your requirements.
Darts on every Wednesday night. Starting 6pm. Prizes each night. $10 to enter.
Also check out our Facebook Page for other activities.
Full Membership entitles you to access all of the club's facilities and all events.
Business House Bowls is run over the summer months. See brochure in upcoming events for full details. Open to non members as well as members. This is your chance to try bowling in a fun social event.
Boxing Day Tournament. Start planning your triples teams now. More information to come.