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UofL Department of Anthropology News & Events
Immigration Clinic
For any current students concerned about immigration status and issues, on February 28, 6:30-9:00 p.m. the Brandeis Human Rights Advocacy Program is hosting an immigration clinic Local attorneys are meeting pro bono with any students, faculty, or staff who may have questions about immigration status. It will be held at the Law School. RSVP is https://www.eventbrite.com/hrap-event-tickets-31700719706.
10th annual Social Justice Research Paper Awards
Last year, one of own, Tyler Short (MA 16) won the Graduate Award.
10th annual Social Justice Research Paper Awards
The University of Louisville's Anne Braden Institute for Social Justice Research is sponsoring its 10th annual Social Justice Research Paper Awards. Students from any discipline are encouraged to apply.
$300 for best graduate essay
$100 each for two best undergraduate essays
· Engage one or more social justice topics. Preference given to papers engaging race,
class, gender, sexuality, religion, peace/conflict, environmentalism, disability, and/or age. Papers do NOT have to be about Anne Braden.
· Undergraduates must use at least one source from the Braden Institute reading room and/or the Braden Papers in University Archives.
· Graduate students should use books and paper collections if relevant to the research topic.
· Paper must be 8-25 pages in length. This is not including reference pages. It must be double-spaced and in 12 point font.
Deadline to submit papers:
Tuesday, May 2 at 4:00 p.m.
Ekstrom Library, Room 258
Winning papers announced:
Friday, May 12, 2017
Visit louisville.edu/braden for full guidelines
Field Course in the Brazilian Amazon- Indigenous peoples, Conservation, and Development
Applications for this summer's University of Maryland study abroad program, Conservation and Indigenous Peoples, are now open! This field course explores the relationship between indigenous peoples, development, and conservation in Brazil's Amazon forest from July 17th - August 3rd.
This is a rare opportunity to explore, learn and work in the Brazilian Amazon with the Kayapó. The course touches on a broad range of topics from anthropology to conservation biology to international education. You can find out more about the program on our course web page.
You can begin your application here. Applications are due March 1, 2017. Your application must be complete by Feb.15th to be considered for a UMD scholarship.
In addition to the incredible learning opportunity, this six credit course meets the UMD General Education Requirements for Scholarship in Practice and Cultural Competence and as well as ECEV enrichment requirement for BSCI students.
There are limited spots on the program, so completing an application early is highly advised, this will make sure your application will be considered.
Interested students can email us directly: Janet Chernela (email@example.com), Matt Aruch email@example.com, or Emily Colon at firstname.lastname@example.com
Feel free to contact us with any questions or concerns!
AAA Calls for Immediate Reversal of Executive Order Banning Immigrants
January 29, 2017
Contact Name: Jeff Martin
Contact Email: firstname.lastname@example.com
AAA Calls for Immediate Reversal of Executive Order Banning Immigrants
The American Anthropological Association (AAA) calls on the US government to immediately reverse its ill-informed, heavy-handed executive order suspending entry of all refugees to the United States for 120 days, barring Syrian refugees indefinitely, and blocking entry into the United States for 90 days for citizens of seven predominantly Muslim countries: Iran, Iraq, Libya, Somalia, Sudan, Syria and Yemen. The Department of Homeland Security immediately began enforcing the order, including barring green card holders from those countries from re-entering the United States, with exceptions to be evaluated on a bureaucratically slow and inefficient case-by-case basis.
"The order must be rescinded, immediately, and the hateful cultural ignorance behind it must be named," said AAA President Alisse Waterston.
Anthropology is dedicated to making the world safe for cultural differences. Our scholarship helps advance understanding by taking the long and comparative view of human behavior. From our perspective, this executive order serves no useful purpose and dramatically reverses decades of precedent without any apparent careful consideration for the violation of human rights of refugees and immigrants and of the prospect for potentially dangerous consequences. This executive order places scholars, students and practitioners in anthropology and other disciplines in serious jeopardy.
"We will continue to muster anthropological knowledge and expertise in the service of upholding human rights, protecting academic freedom and helping solve—not exacerbate—human problems," Waterston added. "To our friends and colleagues around the world, we feel compelled to emphasize this new administration's actions do not reflect the views of a majority or even a plurality of the American people. This government cannot hide behind the spectacle and highly charged xenophobic rhetoric of nationalism any longer. Its policies and practices must be based on knowledge gained from systematic observation. To do otherwise places human rights and the rule of law in peril. We are watching closely, and hold this new administration accountable for remaining within the guide rails of truth and justice."
Founded in 1902, the American Anthropological Association, with 10,000 members, is the world's largest scholarly and professional organization of anthropologists. The Association is dedicated to advancing human understanding and applying this understanding to the world's most pressing problems.
June 1 - July 1, 2017
6 credits (Ethnographic Field School / Language and Culture)
Appalachian State University, UNC system
Director: Dr. Jon Carter, email@example.com and Dr. Christina Sornito, firstname.lastname@example.com
$3,400 (includes: roundtrip airfare from Charlotte to Quito, in-country transportation, food and lodging for 30 days, entrance
Deadline to Apply: February 15, 2017
NOTE: This program is limited to 12 spots with a pre-approval process.
Now in its tenth year, and in collaboration with the Appalachian State University Ethnography Lab (Department of Anthropology), this program will give students the opportunity to travel to Ecuador where they will learn about indigenous culture and language by working with a community in the Amazon. The majority of the program will be spent on the shores of the Napo River, one of the main tributaries that create the Amazon River.
This is an anthropological-based program in which students will take two courses. In the first, Ethnographic Field School, students learn the scope of ethnographic methods used by fieldworking anthropologists, and begin to design small-scale research projects of their own. Students work directly with an indigenous women's cooperative focused on gender, sustainable community tourism, and the transformation/conservation of local traditions. Students work with community members to learn about indigenous representation in Ecuador, and the impact of oil, eco-tourism, and rainforest management on identity, gender, and community empowerment among Kichwa (Quichua)-speakers of the upper Amazon. The program focuses on engaged anthropology and collaborative partnerships between scholars, students, and local community members. For the second course, Language and Culture, students learn about the Kichwa language, and the politics of language preservation, heritage, and cultural activism in Ecuador.
Dr. Jon Carter and Dr. Christina Sornito, field school directors and co-directors of the Appalachian State Ethnography Lab, work with students to produce final projects that draw from readings in ethnographic writing, visual anthropology, ethnographic film, the anthropology of sound, and sensory ethnography. Students work in collaboration with indigenous and community organizers to produce ethnographic works (final projects) on human and animal worlds, environmental politics, history, resource extraction, cultural identity, gender, tourism, and other themes that emerge from course readings. Working together, students will develop their own research goals and carry out their research with local community members.
Students will visit family homes with indigenous translators and guides, to meet and spend time with local families, and receive an introduction to local traditions and practices in the way of manioc
(cassava/yuca) harvesting, community service projects (mingas), ceramic traditions, environmental conservation, and "shamanism".
Students also hike into the Amazon jungle, where they will learn about local plants used for medicinal purposes and regional biodiversity.
Students have come from Pennsylvania State University, Cornell University, Indiana University, York University, Tufts University, Louisiana State University, Bowdoin College, Eckerd College, St.
John's College, University of New Mexico, University of Alabama, University of Illinois-Chicago, University of North Carolina at Chapel Hill, Florida International University, and many others. Their majors have included anthropology, linguistics, geography, biology, global studies, political science, women's studies, global health, sustainable development, interdisciplinary studies, social work, sociology, and studio art.
Alumni of this program have been accepted to graduate programs (i.e.
the University of Michigan, Arizona State University, University of Chicago, London School of Economics and Political Science, Wake Forest University, Arizona State University, the University of Denver, and the University at Albany-SUNY), while others have used this experience to land internships and work with NGOs after graduation such as The Carter Center for Human Rights, AmeriCorps, Language Development and Perception Laboratory at the University of Maryland, Clinton Global Initiative, Université de Lausanne, Yellowstone National Park, Threads of Peru, Cornell University BABY Lab, North Carolina One Health Collaborative, and Latino Health Program of the High Country
For more information on the program and how to apply, please copy and paste the following link into your web browser (in case hyperlink does not work): https://urldefense.proofpoint.com/v2/url?u=https-3A__anthro.appstate.edu_field-2Dschools_ethnographic-2Dand-2Dlinguistic-2Dfield-2Dschools_summer-2D2017-2Decuador&d=AwIGaQ&c=SgMrq23dbjbGX6e0ZsSHgEZX6A4IAf1SO3AJ2bNrHlk&r=BijBVFt7inteN1QtyyzsQUH3eEFh0lkHl3-Wq3dtkXo&m=IOeyhqaUVVpe9AnXMufmcIUtzYKPd-8VTfhD9Z89cQU&s=GmXt2-B-MBvSepJkCNuH08b0jDIoPX1EImU0MPeytgs&e=
We encourage anyone who is interested to contact the program co-director, Dr. Jon Carter, for further information, at email@example.com
American Anthropological Association Post-Election Statement/Resolution
The American Anthropological Association reaffirms our fundamental commitments to academic freedom, human rights, a respect for all persons, and advancing our collective understanding of the human condition as a path towards a more just and sustainable world. We were fortunate to be able to come together as an association of scholars, students, and professionals at our Annual Meeting in Minneapolis in the week after this month's US national elections to reaffirm our shared ideals and chart a way forward. In accordance with our Association's governance rules, our Executive Board listened carefully to advisory motions presented at the Annual Business Meeting, and responded by approving the first proposed AAA resolution. In addition, this resolution has been endorsed unanimously by all of the living past Association presidents.
A Resolution on Behalf of the American Anthropological Association in the Wake of the 2016 National Elections
Whereas, The 2016 national election campaign season has been characterized by painfully divisive, often threatening rhetoric regarded by many as racist, anti-immigrant, misogynistic speech that has touched every corner of American society and, indeed, the entire world;
Whereas, Organizations advocating racism, gun violence, and misogyny openly endorsed the winning presidential candidate;
Whereas, During the course of the 2016 campaign, numerous threats were made against anti-racist and feminist politicians, academics, journalists, and activists;
Whereas, A spike in hate crimes and harassment has followed the election, with more than 700 reported accounts of hate crimes in the US in the weeks since the election results were announced;
Whereas, The discipline of anthropology is distinctively placed to contribute valuable insights to advance our collective understanding of migration, cultural diversity, and racism;
therefore, be it
RESOLVED, That the American Anthropological Association rejects in the strongest terms the climate of hostility that threatens the personal and intellectual diversity of American society;
RESOLVED, That the Association reaffirms its commitment to protecting the pursuit of free inquiry about the human condition with scholarly rigor, offering the greatest possible opportunity for people to take part in and benefit from that inquiry, and engage the many communities that make up the United States and the world in valuing diversity;
RESOLVED, That the Association urges its members to stand in solidarity with students, colleagues, employees, and community-based collaborators who feel they are threatened or under attack, and engage with local civic organizations to effect positive outcomes in their own communities;
RESOLVED, That the Association is dedicated to working collaboratively with other scholarly and professional organizations and institutions of higher learning to honor its commitments, to monitor, intervene, and update its membership on key issues that have a clear impact on anthropology, and to participate as a valued disciplinary stakeholder in shaping policy outcomes rooted in core values of mutual respect, equal rights, freedom of expression and freedom from discrimination.
Founded in 1902, the American Anthropological Association, with 10,000 members, is the world's largest professional organization of anthropologists. The Association is dedicated to advancing human understanding and addressing the world's most pressing problems.
The Smithsonian Office of Fellowships and Internships
Programs now accepting applications include:
The Smithsonian Institution Fellowship Program (for Graduate Students, Predoc Students, Postdoc Researchers, and Senior Researchers)
The Smithsonian Biodiversity Genomics Postdoctoral Fellowship
The Smithsonian Postgraduate Fellowship in Conservation of Museum Collections
The Smithsonian Artist Research Fellowship (SARF)
The Smithsonian Mpala Postdoctoral Fellowship
Please note the deadline for these programs has changed from last year. The new application deadline for most of these programs is now Thursday, December 1, 2016.
For more information, go to http://smithsonianofi.com or call the Smithsonian Office of Fellowships and Internships at 202-633-7070.
470 L'Enfant Plaza SW Suite 7102
Washington, DC 20013-7012
Political Ecology Working Group
The Political Ecology Working Group (PEWG) is an interdisciplinary group of graduate students at the University of Kentucky, who organize and host the annual Dimensions of Political Ecology (DOPE) Conference. Since its inception in 2010, this student-organized conference has become one of the largest, most highly-regarded international forums for critical discussions at the intersection of ecology, political economy, and science studies. DOPE 2016 welcomed more than 300 scholars, environmental professionals, and activists from over 100 institutions to Lexington. The DOPE Conference has offered a platform for both established and emerging scholars, featuring invited speakers such as Paul Robbins, Rebecca Lave, Erik Swyngedouw, Vandana Shiva, Julie Guthman, Laura Pulido, and Kim Tallbear.
For more information, visit http://www.politicalecology.org
Congratulations to Spring and Summer M.A. Anthropology Graduates
Congratulations to Tyler Short, Amanda Wharfield, and Neha Angal for successfully completing their M.A. degrees. Tyler defended his thesis 'La Minga as a model of food justice?: a thesis on the motivations and practices of immigrant and native-citizen growers at La Minga cooperative farm in Prospect, Ky' and graduated in May 2016. Amanda completed her internship at Catholic Refugee Ministries and presented her internship report and graduated in May 2016. Neha defended her thesis 'Effects of Migration, Carrying Capacity, and Fecundity on the Formation of Clinal Patterns during Range Expansions' and will graduate in August.
Several Anthropology Undergraduate and Graudate Students Present and Attend 2016 AAPAs
Several anthropology students attended and presented at the 2016 American Association of Physical Anthropology meetings in Atlanta. Undergraduate students Cori Dennison and Megan Duncanson presented research conducted under their mentor, Dr. Fabian Crespo. Dr. Crespo presented work completed with graduate student Chris Klaes. Graduate students Roxanne Leiter and Neha Angal presented segments of their thesis research conducted under their mentor, Dr. Christopher Tillquist. Lastly, graduate students Mallory Cox and Austin Warren attended the meetings to network and enjoy their first professional meetings.
"This year at the Annual Association of Physical Anthropologist's meeting I got the opportunity to present a poster with my preliminary thesis research. I was also able to attend symposiums about current and future directions of anthropological research. The highlight of this year's meeting was having the opportunity to discuss my thesis research with an anthropological geneticist whose previous work has been pivotal in my research. The annual meetings are particularly valuable when students have the chance to talk one on one with researchers that are or have been involved in their area of specialization. These meetings are a great opportunity to network, make improvements to our research and get good practice in presenting our research clearly and effectively."
-Roxanne Leiter, graduate student
-Cori Dennison, undergraduate student
"Attending and presenting at this year's AAPA's was an awesome experience! It was so cool to have the opportunity to share my work with other students and researchers and to get valuable feedback on a portion of my thesis research. The meetings were also great for networking and staying current on the latest research trends and directions, and for reconnecting with friends from other universities. Presentations on topics such as ancient genomes and micro biomes, adaptive introgressions in the Homo lineage, and innovative studies combining ancient DNA, collaborative work with indigenous communities, and the robusticity of simulations and complex modeling were particularly thought provoking!"
-Neha Angal, graduate student
"This semester I had the opportunity to travel to Atlanta for the annual Paleopathology Association conference, as well as the annual meeting of the American Association of Physical Anthropologists. These meetings bring together some of the leading experts in a variety of fields related to physical anthropology and provide an invaluable experience for students like myself. I was able to present a poster of my ongoing project with Dr. Crespo at the PPA meeting, and had the opportunity to receive feedback from a variety of students and professors about our work. While it can be intimidating to talk one on one with big names in the field, it proved to be an excellent learning experience. For the rest of the meetings I was able to enjoy the talks and presentations by leading researchers, and especially enjoyed seeing the work of fellow U of L anthropology students. The conference was very productive this year, and I am excited for what the 2017 meetings have to offer."
-Megan Duncanson, undergraduate student
Graduate Students Brandon Zinsious and Melissa Holst Present at the 2016 SAAs
Participating in this years SAAs allowed me to present my research (in the form of a poster) for the first time to a professional audience. During this presentation I was not only offered invaluable feedback and encouragement on my research endeavors, but I was also able to network with members of the archaeological community that I may not have had the opportunity to approach on my own. Outside of my presentation I was also able to attend numerous types of other presentations, lectures, and expos. All of these various presentation mediums provided new or further insight into various archaeological topics or techniques and provided further networking opportunities. I feel that from this conference I not only learned more about the up and coming research and researchers in the field, but I learned a lot about my own research and feel encouraged now more than ever to continue doing what I do.
-Melissa Holst
LL.M. Program in Agricultural & Food Law Fall 2016 Applications Still Open
The LL.M. Program in Agricultural & Food Law continues to accept applications for Fall 2016, with a few places remaining in face-to-face and distance tracks, for full or part-time enrollment. With an expanded curriculum and a deep base of alumni relationships, their program prepares attorneys for a career in agricultural and food law. Visit the website and blog for additional information.
They have several Graduate Assistantships (GAs) to award. GAs are only available to full time LL.M. candidates who enroll in the face-to-face program. These GAs provide for a full tuition waiver plus a $5,000 stipend per semester in exchange for part-time work designed to enhance their education and build their professional reputation. While awards may shift to accommodate the expertise of applicants, GA placements are likely to include:
- An opportunity to work with Accelerated JD candidates from foreign jurisdictions, assisting them with their transition to a U.S. law school setting;
- An opportunity to teach a Pre-Law Political Science class that introduces undergraduates to basic elements of our legal system and encourages them to explore a legal education:
- An opportunity to teach an Upper Level Legal Writing class that focuses on Civil Pre-Trial documents (opportunity limited to attorneys with practice experience and/or LRW teaching experience);
- Opportunities to assist the LL.M. Program or the Indigenous Food & Agriculture Initiative with a variety of projects including research, writing, and resource development.
Interested attorneys and graduating 3Ls should complete the LL.M. application and indicate their interest in one or more of the GA opportunities. Awards are highly competitive. Contact us for additional information at firstname.lastname@example.com or call (479) 575-3706.
It is always the goal of the LL.M. Program to attract candidates that reflect the rich racial, cultural, ethnic, and geographic diversity of a global food system, expanding the reach and resources to all who seek to promote food justice.
Two AmeriCorps VISTA positions for Food Literacy Now Available
Applications are due May 4th. Please refer to the flyers below for all details:
AAA Graduate Student Human Rights Paper Award
Call for Papers: Guerrero-Friedlander Human Rights Graduate Student Paper Prize
The American Anthropological Association Committee for Human Rights (CfHR) invites graduate students to apply for the Guerrero-Friedlander Human Rights Graduate Student Paper Prize. Eligible students are enrolled in a graduate degree-granting program until at least May 1, 2016. Eligible programs include, but are not limited to JD, LLM, MS, MA, MPH, MS, or PhD.
Papers should demonstrate an outstanding understanding of and critical engagement with human rights issues from an anthropological perspective, addressing both theory and practice. Topics of research are not geographically restricted and may include, but are not limited to: colonialism, civil rights, displacement and migration, environmental rights, genocide, health disparities, Indigenous rights, land claims, language and justice, nativism, peace and conflict, political economies of human rights, refugees and asylum seekers, social justice, social movements, sovereignty, structural and systemic violence, and warfare. Submissions that follow a participatory action approach or emphasize anthropological action/practice are encouraged.
The CfHR awards a cash prize of $400 which will be presented at the 2016 American Anthropological Association Annual Meeting during the Committee for Human Rights Public Forum, and the student will be asked to provide a 15-20 minute presentation on the winning paper at this time. The winner will also be profiled in Anthropology News.
Submissions should not exceed 8,000 words (including notes and references) and should follow AAA style guidelines: www.aaanet.org/publications/style_guide.pdf<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.aaanet.org_publications_style-5Fguide.pdf&d=AwIF-g&c=SgMrq23dbjbGX6e0ZsSHgEZX6A4IAf1SO3AJ2bNrHlk&r=BijBVFt7inteN1QtyyzsQUH3eEFh0lkHl3-Wq3dtkXo&m=FdHWdcSsyqqDd-eO--QfQoJ2Ce2FDMavkl8Sh1rp0TM&s=2UUBtrC3CgBvckTagTdkGrtMXXxkDUShi86IMX2eNuk&e= >. Submissions should be unpublished manuscripts not currently under review elsewhere, forthcoming, or in press. Please submit two pdfs for review to Jaymelee Kim at email@example.com<mailto:firstname.lastname@example.com> by September 1, 2016.One version should be complete: including a cover sheet with the author's name, contact information, paper title, abstract, and keywords that accompanies the paper; the other version should include the paper's title, text, and abstract, but no identifying information. Papers will be read in a double-blind process by Committee for Human Rights members.
Christian Brawner, Anthropology Dept Undergraduate Student, Profiled by The Louisville Cardinal
Christian Brawner--Demonstrating the power of activism
Here's a wonderful profile written by Eiman Zuberi for the Louisville Cardinal of Honors program senior Christian Brawner. He is the only student to win both a 2016-2017 Fulbright (ETA Jordan), and a CLS summer placement (Advanced Arabic, Morocco).
Follow this link to read the article: http://www.louisvillecardinal.com/2016/04/demonstrating-the-power-of-activism/
Field Study in Peru Fall 2016
Study abroad in Tarapoto, Peru Fall 2016!
If you are interested in a study abroad experience, hiking in the Amazon region, and Peruvian culture, then the Fall Course Comm 510-01: Field Study in Peru is for you. Please contact: firstname.lastname@example.com for details.


text: 24 photos $310,000 17.44 acres 4 Beds | 2 Baths | 3376 sqft 1485 Cummins Ferry Road, Salvisa, KY 40372 - Mercer County 17.44 acres Salvisa, KY Pleasure Horse Operation located in North Mercer. Convenient to Versailles, Lexington, and Harrodsburg. This farm sits on 17.44 slightly rolling acres. This property consist of a modern 4 bedroom 2 bath, brick home with a full finished basement. A three car attached garage with storage. Large tobacco barn with water and electric presently stalled for 8 horses with ample room for more stalls and riding. Lots of room for outside training. This property has good fencing with 3 separate paddocks and room for two more with added fencing. There is also a block building with concrete floor multi use. Must see to appreciate. Ritchie McGinnis RM McGINNIS, LLC Real Estate & Auction Professionals

subset: commoncrawl


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With the Epson Stylus Pro 3880, uncompromising quality is within reach. Engineered for the discriminating professional, this compact, 17"-wide printer includes Epson UltraChrome K3 with Vivid Magenta Ink Technology, world-renowned for professional printing applications. This, combined with an advanced MicroPiezo AMC print head, enables the Pro 3880 to produce gallery-quality color and black-and-white output up to 17" x 22".
The printer's industry-acclaimed ink set delivers a phenomenal color gamut with breathtaking blues and violets. Now, you can achieve stunning wide-format prints to professionally showcase your work and bring your creative visions to life.
The Epson 3880 is commonly used for Photos and more.The Epson 3880 is most used by customers who consider themselves to be a Frequent user among others.The Epson 3880 is popular because customers like the following qualities of the Epson 3880: Compact, Easy to set up, Easy to use, Fast print time and Reliable.
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text:
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text:
"Let me tell you something, being an artist means that I'm extremely good with my hands and...fingers..." He held her wrists tightly above her head, chest against chest he felt her heart beating rapidly.
"...and I plan on showing you just how good."
Grace is just your average girl that didn't expect a clumsy mistake to change her life. All her life she's been struggling to get where she wants to be so when an obstacle named Noah Miller appeared she simply saw him as a challenge which unexpectedly turned the devil himself into a blessing.
When Grace ruined Noah's most prized possession he made sure she'd pay for it, he never meant for their agreement to turn into his craving.
MusicIsWriting_06 Oct 18, 2016
Oh honey. So if I paint a pretty picture of an apricot and a middle finger, will I earn $35,000 for my beautiful masterpiece? ;) 💞💞
MusicIsWriting_06 Oct 18, 2016
A thumb on an orange? Is there a hidden joke behind this? (: Maybe I'm just too bland to get it..? Haha💞
Nicky_Oden Nov 16, 2016
Potatoes are amazing i mean they make vodka from potatoes so how can it not be amazing
Beautiful_Starz Oct 19, 2016
*smacks forehead* 🙄😯 oops, my bad bro! Let me just grab my wal- wait what? $35,000?!! Well I guess you want monopoly money. 😒Cuz that's the only way you're about to get that amount from me.
HerKaleidoscopeEyes Feb 03
I try but can never seem to really understand modern abstract art. Now, impressionist and expressionist I get and like.
KeepStayingWeird Nov 26, 2016
Like he shouldn't have done that but still she did ruin his painting


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Would you like to land a prestigious, four-year paid apprenticeship, build your career, and make a difference in the world while pursuing your associate degree at New England Tech? If you plan to pursue a degree in one of NEIT's building trades associate degree programs, you are eligible to apply for a paid apprenticeship through the Naval Facilities Engineering Command (NAVFAC) apprenticeship program at the Navy bases in either Newport, RI or Groton, CT.
Join us on either Tuesday, December 13 or Wednesday, December 14 anytime between 4:30 – 6:00 pm, on our main campus, One New England Tech Boulevard, East Greenwich RI, 02818. You'll get to meet with a senior representative from NAVFAC who will discuss the apprenticeship program in greater detail and answer any questions you might have.
If you qualify and are accepted into the program, you'll work alongside NAVFAC personnel mentors who are the Navy's installation facility experts.
Through this experience, you'll build your résumé while providing tangible value to our nation's defense and our citizens.
You'll be eligible to apply for an apprenticeship if you enroll in any of NEIT's associate degree programs in electrical technology, or heating, plumbing, or refrigeration technology.
Just announced – NAVFAC presently has vacancies and will consider candidates from NEIT's automotive technology program to fulfill apprenticeship duties as a heavy mobile equipment mechanic.
Military veterans are especially encouraged to apply. The Federal Government has a long and outstanding record of employing veterans. Veterans have technical skills in areas of critical importance, and many already have security clearances required for some Federal positions. Federal service provides a means for veterans to continue their noble service to this country while pursuing their advanced degree.
For more information about this apprenticeship opportunity – and about your New England Tech degree program – contact Admissions at 401-467-7744 or email us at firstname.lastname@example.com. We'll be more than happy to sit with you and explain all your options.


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About 60 people have participated in a walk against bullying in the Tamworth CBD.
Topics: bullying, mental-health, tamworth-2340
Topics: bullying, mental-health, tamworth-2340
The Mayor of North Sydney is using new federal laws to tackle workplace bullying against a fellow councillor.
Topics: bullying, work, local-government, north-sydney-2060
Posted| Updated
A campaign is encouraging people to avoid using social media for 48 hours this weekend in a bid to raise awareness and funds to fight cyber-bullying.
Posted| Updated
An Australian organisation that works with victims of bullying is calling on teenagers to participate in a 'digital detox' this weekend.
Topics: social-media, bullying, schools, australia, melbourne-3000
Posted| Updated
Australian teenagers are being asked to completely disconnect from all forms of social media for 48 hours as part of a 'digital detox' campaign to mark National Day of Action Against Bullying.
Topics: internet-culture, social-media, bullying, melbourne-3000, australia
Posted| Updated
Teachers are spending a significant amount of class time dealing with problems related to weekend alcohol and drug abuse, according to the results of a national survey.
Topics: youth, drugs-and-substance-abuse, community-and-society, mental-health, health, bullying, schools, teachers, education, alcohol, drug-use, australia
Posted| Updated
When I began my career as a television journalist, I had no idea just how helpless you can feel when being attacked by someone who gives you no room for reply.
Topics: media, bullying, social-media
Posted| Updated
The death of Charlotte Dawson has Australia reflecting on depression and the increasing incidence of cyberbullying via social media websites.
Topics: social-media, suicide, information-and-communication, internet-culture, bullying, woolloomooloo-2011, australia
Posted| Updated
South Koreans will have the option to insure themselves against the school bully.
Topics: bullying, insurance, korea-republic-of, asia
Posted| Updated
Anti-bullying campaigner John Caldwell is Victorian of the Year.
Posted| Updated
The sister of a 17-year-old boy who committed suicide after being bullied has made a statement at the inquest into his death.
Topics: suicide, courts-and-trials, bullying, bathurst-2795, sydney-2000
Posted| Updated
New research has found the stress of working in busy commercial kitchens is driving apprentice chefs to drink.
Topics: education, alcohol-education, drug-use, health, alcohol, business-economics-and-finance, industry, hospitality, bullying, australia, qld, brisbane-4000
Posted| Updated
The mother of a Geelong teenager who took her life in 2009 believes reports about other suicides in the media contributed to her daughter's death.
The Coroners Court is sitting in Geelong to examine the deaths of teenagers Chanelle Rae, Zach Harvey and Taylor Janssen, who killed themselves in 2009.
Topics: suicide, adolescent-health, courts-and-trials, bullying, social-media, media, secondary-schools, geelong-3220
Parents of school age children are invited to attend a free anti-bullying seminar at the University of Newcastle today.
Topics: bullying, newcastle-2300
Posted| Updated
The Department of Education says it stands by a southern Queensland school's decision to suspend 39 students over a fight last week.
Topics: public-schools, secondary-schools, bullying, centenary-heights-4350, toowoomba-4350
Posted| Updated
St Patrick's College in Campbelltown has lost its appeal against a former pupil's $100k compensation claim.
Topics: bullying, courts-and-trials, schools, campbelltown-2560
Catholic Education in Townsville in north Queensland has issued warning letters to parents about an increase in the use of social media for bullying.
Topics: bullying, parenting, family-and-children, internet-culture, townsville-4810
Posted| Updated
The ABC's Deborah Rice is working on a story about young women who have recovered from self-harm and can suggest positive strategies to help others.
Topics: youth, community-and-society, family-and-children, grief, bullying, information-and-communication, social-media, australia
Posted| Updated
An independent investigation into allegations of workplace bullying at the CSIRO has found no major or widespread issues.
Topics: work, bullying, australia, canberra-2600, act
An outback Queensland mayor says he is concerned residents could leave town if problems are not dealt with at Birdsville State School in the state's far south-west.
Topics: public-schools, public-sector, bullying, birdsville-4482, longreach-4730
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Parents are refusing to send their children to the Birdsville State School in outback Queensland today over bullying allegations.
Topics: public-schools, bullying, community-development, regional, birdsville-4482, longreach-4730, mount-isa-4825
Posted| Updated
A former chief justice of the Family Court has called on the Federal Government to consider making cyber bullying an offence punishable by jail time.
Topics: bullying, internet-culture, laws, family-law, law-crime-and-justice, australia
Posted| Updated
A conference in Melbourne is discussing the possible development of anti-bullying laws for Australia.
Topics: bullying, internet-culture, social-media, australia
Former Chief Justice of the family court, Alastair Nicholson, discusses the upcoming symposium to reconsider ways to fight bullying.


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Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.
Methodology/Principal Findings
We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.
Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.
Citation: Bocchino M, Agnese S, Fagone E, Svegliati S, Grieco D, Vancheri C, et al. (2010) Reactive Oxygen Species Are Required for Maintenance and Differentiation of Primary Lung Fibroblasts in Idiopathic Pulmonary Fibrosis. PLoS ONE 5(11): e14003. doi:10.1371/journal.pone.0014003
Editor: Melanie Koenigshoff, Comprehensive Pneumology Center, Germany
Received: February 10, 2010; Accepted: October 18, 2010; Published: November 16, 2010
Copyright: © 2010 Bocchino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by Associazione Italiana per la Lotta al Cancro (A.I.R.C.) and by a generous grant from Fondazione Cariverona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disorder with a mean survival of 3–6 years from the onset of symptoms. Histology of IPF shows the features of usual interstitial pneumonia with patchy distribution of fibrosis adjacent to fibroblastic foci (FF) [1]. IPF appears to be an "epithelial-fibroblastic disease" resulting from recurrent epithelial injury and abnormal wound repair [2]. FF are composed of migrating and proliferating fibroblasts and of differentiated myofibroblasts accounting for extra-cellular matrix deposition slowly altering the alveolus structure. This explains the progressive and irreversible IPF nature and the prognostic value of the fibrosis extent [3], [5].
IPF pathogenesis is unknown and the role of inflammation remains controversial, since anti-inflammatory treatment does not produce significant benefit against the disease progression. Inflammation is likely the triggering event for the initiation of fibrosis; eventually, fibrosis self-maintains and progresses by an unknown process [6], [7]. Recent studies have emphasized the role of oxidative stress as the molecular basis of lung fibrosis. Reactive oxygen species (ROS) are key players in the establishment/progression of pulmonary fibrosis in animal models and possibly in human IPF [8]–[11]. There is evidence of disruption of the normal oxidant/antioxidant balance in the lungs of IPF patients. Deficiency of antioxidants, including glutathione and superoxide dismutase, has been found in the lower respiratory tract of IPF patients, while high levels of myeloperoxidase are associated with epithelial injury in the fibrotic lesions [12]–[14].
Fibroblasts and myofibroblasts are recognized as the effector cells in normal wound healing and in the development of tissue fibrosis [15]. Although the interaction of these cells with a large spectrum of growth factors involved in tissue remodelling has been extensively investigated in IPF, their relationship with oxidative stress remains poorly clarified. The aim of the present study was to characterize the baseline cellular phenotype of fibroblasts derived from IPF patients and to identify molecular targets underlying this phenotype.
Materials and Methods
Ethics Statement
The study was approved by the Institutional Review Board for biomedical activities of the Universities of Naples, Ancona and Catania and by the Ethics Committee of the Monaldi hospital, Naples, all in Italy. Patients provided written informed consent.
Cell culture
Primary lines of human lung fibroblasts were established by using an outgrowth from explant following the method described by Jordana et al. [16]. IPF cell lines were obtained from 7 patients affected by IPF (age range 48–60 y), undergoing surgical lung biopsy for diagnosis. Control fibroblasts were derived from normal lung tissue of 4 patients with tumour-free areas of lung lobes with early stage bronchial carcinoma (age range 45–55 y). Cells were grown under standard conditions at 37°C in 5% CO2 in DMEM with 1 g/l glucose supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin, and used at 80–90% confluence at different culture passages. To distinguish experiments with different culture timing, cells used within passage VI were arbitrary referred as "early passage" while cells used later on through passages IX-XI were referred as "late passage". Overall, early passage cells were defined as cells within 20–25 population doublings (PD), while late passage cells had a PD greater than 45. All reagents were purchased from GIBCO (Scotland, UK). All experiments were performed in duplicate.
Cell treatments
Reagents used for cell treatments included: 1. H2O2 (Sigma, St. Louis, MO, USA); 2. the recombinant human transforming growth factor (rhTGF)-β1 (Calbiochem, Merck Darmstadt, Germany); 3. the TGF-β receptor ALK5 inhibitor SB-431542 (Sigma); 4. the ROS scavenger, N-acetylcisteine (NAC, Sigma); and 5. the general oxidase inhibitor diphenyleneiodonium (DPI, Calbiochem). Additional treatments included the MEK kinase inhibitor UO126 and the receptor tyrosine kinase (RTKs) inhibitors AG1296 (PDGF-R inhibitor) and AG1478 (EGF-R inhibitor) (Calbiochem) [17]. Dose-response curves were performed with all reagents.
Cell proliferation assay
Proliferation assays were performed in 96-well microtiter plates. Cells were cultured at a density of 1×103 viable cells per well for 48 h and pulsed for additional 24 h with 0.25 µCi/well of [3H] thymidine (Amersham, Pharmacia Biotech Buckinghamshire, UK). Cells were harvested on glass-fiber filters using a Tomtec (Orange) 96-well cell harvester and counted in a 1205 Betaplate liquid scintillation counter (Wallac). Results obtained from three wells were expressed as mean cpm±SD.
Cell death assay
Non adherent and trypsin-treated adherent cells were collected and washed twice with cold PBS. Cell pellets were re-suspended in 1 ml of propidium iodide (PI) staining solution (PI 5 µg/ml). Samples were kept at room temperature (RT) in the dark and immediately analysed by flow cytometry (Cyan DAKO Cytomation, Colorado, USA). For each sample 30.000 events were acquired and PI positive cells were detected at a wavelength of 635 nm. Data analysis was carried out with the Summit software (version 4.3, DAKO).
Senescence β-galactosidase assay
Medium was removed from sub-confluent cultures. Cells were fixed with 3% formaldehyde for 10 min at RT, washed and incubated overnight at 37°C in staining solution containing 1 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), purchased from Promega (Madison, WI, USA), 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2. Cells were checked under a microscope for development of blue colour and the mean percentage of β-galactosidase-positive cells was calculated by counting 3 representative fields.
RNA purification and real-time PCR
Total RNA was extracted with TRIzol reagent according to the manufacturer's protocol (Sigma) and two micrograms were reverse-transcribed with the Omniscript RT kit (Qiagen, Milan, Italy) using random primers (1 µM) at 37°C for 1 h. Real time PCR was performed in triplicate in 20 µl reaction volumes using the Power SYBER Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCR primers (0.2 µM) for human A2 type-I collagen used were: sense 5′-TCTGGAGAGGCTGGTACTGC-3′ and anti-sense 5′-GAGCACCAAGAAGACCCTGA-3′. PCR primers (2 µM) for human transforming growth factor (TGF)-β used were: sense 5′-ACTACTACGCCAAGGAGGTCAC-3′, anti sense 5′- TGCTTGAACTTGTCATAGATTTCG-3′. 18S RNA was used as reference (sense 5′-TCC CCA TGA ACG AGG AAT TC-3′ and anti-sense 5′-GTG TAC AAA GGG CAG GGA CTT-3′). All primers were purchased from Invitrogen (CA, USA). Real time PCR reactions were carried out in a MJ Mini™ Personal Thermal Cycler apparatus (Bio-Rad Laboratories, CA, USA). Melting curves were obtained by increasing the temperature from 60 to 95°C with a temperature transition rate of 0.5°C/sec. The comparative threshold cycle number (CT) method was used to assess the relative quantification of gene expression. The fold change of the target gene was calculated as 2−ΔΔ CT [18].
Determination of reactive oxygen species (ROS)
Fluorimetric determination of intracellular ROS generated by adherent cells was performed in a 24-well plate after loading with 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) (Molecular Probes, Invitrogen, Paisley, UK), as previously described [19]. The intracellular ROS content was also measured by flow cytometry (Cyan). Harvested cells were incubated with 5 µM DCHF-DA for 20 min in the dark at 37°C, washed and re-suspended in PBS. For each sample, 10.000 events were acquired and intracellular ROS formation was detected as a result of the oxidation of DCHF at a wavelength of 520 nm. Data analysis was carried out with the Summit software.
Superoxide anion release was estimated by means of the superoxide dismutase-inhibitable cytocrome c reduction, as previously described [20]. Briefly, 15×103 cells/well were plated in 24-well plates and incubated in Krebs-Ringer phosphate buffer with glucose (145 mM NaCl, 4.8 mM KCl, 0.5 mM CaCl2, 1.2 mM MgSO4, 5.7 mM NaPO4, 5.5 mM glucose [pH 7.4]) (KRPG) containing 80 µM ferricytochrome c (type III; Sigma), with or without SOD (final concentration 300 units/ml), in a total volume of 1 ml. After incubation for 1 h at 37°C, the reaction was stopped by placing the culture supernatants in melting ice, and the absorbance was read at 550 nm in a Sclavo Reader SR400 spectrophotometer (Siena, Italy). An extinction coefficient of 21×103 M−1cm−1 was used for oxidized versus reduced cytochrome c.
Western blot
Harvested cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholate,0.1% sodium-dodecyl-sulfate, SDS) containing 2.5 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 1 mM NaVO4, 1 mM NaF and a cocktail of protease inhibitors (Roche Diagnostic GmbH, Mannheim, Germany) for 30 min at 4°C. Cell lysates were centrifuged at 13.000 rpm for 10 min at 4°C and the concentration of cellular proteins was determined using the Bio-Rad assay (Bio-Rad). Cell proteins (20–30 µg) were mixed with a Laemmli buffer, separated on 12% SDS-PAGE, transferred onto a nitrocellulose membrane (Protan, Whatman GmbH, Dassel, Germany) and hybridized with the following antibodies according to the manufacturer's instructions: mouse anti-α-smooth muscle actin (SMA) mAb (1∶500; Sigma), mouse anti-TGF-β mAb (1∶500; BD Pharmingen, New Jersey, US), mouse anti-p-ERK-1/2 mAb (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-phosphotyrosine (p-Tyr) mAb (1∶1000; Upstate, Lake Placid, NY, USA). Membranes were incubated with an horseradish peroxidase-conjugated anti-mouse secondary antibody (1∶2000; Amersham Pharmacia Biotech) and protein bands were revealed with the enhanced chemiluminescence (ECL) system detection kit (Roche). Immune-reactive bands were quantified by means of densitometry and normalized by mouse anti α-tubulin (1∶3000; Cell Signalling Technologies, Boston, MA, USA) mAb.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). All comparisons were made on a head-to head basis using the paired or unpaired t test, where appropriate. One way repeated measures ANOVA with pot hoc Bonferroni correction was used, where appropriate. A p value less than 0.05 was considered statistically significant. All analyses were performed with the GraphPad Prism 3 software package (GraphPad Software, San Diego, CA, USA).
IPF Cells Display a Pro-Fibrotic Phenotype
To characterize the phenotype of IPF cells we measured the expression of the myofibroblast differentiation marker, α-SMA, and of type-I collagen. Expression of both molecules was increased in early passage IPF cells compared with control fibroblasts (Figure 1, A–B). Alpha-SMA expression increased in control and IPF fibroblasts with time in culture (Figure 1A), while that of type-I collagen was markedly reduced in late passage IPF cells and decreased further in control fibroblasts (Figure 1B). Proliferation of IPF cells, measured as DNA synthesis, was significantly less vigorous than that of control cells at early passages (Figure 1C). Proliferation significantly slowed down with time in culture in both cell types. However, expression of the senescence marker β-galactosidase was only barely detectable in both cell types with no substantial differences even at late passages (Figure 1D). Finally, to address whether the IPF phenotype was sustained by the ability of cells to produce growth factors, i.e. TGF-β, acting in an autocrine loop, α-SMA expression was analysed in early passage control fibroblasts conditioned for 48 h with the complete medium from control or IPF cell cultures. TGF-β is a pleiotropic cytokine that plays a crucial role in a wide array of processes, including wound healing and fibrogenesis. It is stored in the extra-cellular matrix as an inactive molecule which becomes activated after tissue injury. There is evidence that, among others, the integrin-mediated activation of TGF-β is pivotal in IPF pathogenesis [21]. As shown in Figure 2A, no significant differences in α-SMA expression occurred in the culture conditions used. These findings were not unexpected as integrin-mediated mechanisms were unlikely to have occurred in our experimental setting. Further, TGF-β protein levels were undetectable in both cell extracts and culture media from early passage control and IPF fibroblasts (Figure 2B). Also, no differences of TGF-β expression were appreciated at the mRNA level in control and IPF cells, both at early and at late passages (Figure 2C). Finally, sensitivity of early passage IPF fibroblasts to specific TGF-β receptor inhibition was assessed. As shown in Figure 2D, α-SMA expression was unchanged as compared to basal levels.
The baseline phenotype of control (n = 4) and IPF cell lines (n = 7) grown in complete medium was analyzed at different culture passages. Cell lysates from sub-confluent cultures were immune-blotted with anti-α-SMA antibodies. Panel A shows a representative western blot of α-SMA (upper) and tubulin (lower) proteins in control and IPF fibroblasts at early and late passages. Relative statistics for early (full bars) and late (dashed bars) passage cells are also reported. *p<0.05 versus early passage control cells; **p<0.05 versus early passage IPF cells. Panel B shows real time PCR expression levels of type-I collagen mRNA in control and IPF fibroblasts at early (full bars) and late (dashed bars) culture passages. *p<0.001 versus early passage control cells; **p<0.001 versus early passage IPF cells. Panel C shows the proliferation rate of early (full bars) and late (dashed bars) passage control and IPF fibroblasts. *p<0.001 versus early passage control cells; **p<0.001 versus early passage IPF cells. Data are expressed as mean fold change ± SD. Late passage control and IPF fibroblasts stained for senescence associated β-galactosidase activity are shown in panel D (magnification 10X). Arrows show β-galactosidase positive blue cells. The mean percentage of β-galactosidase positive cells ± SD in late passage cells (dashed bars) is reported.
Early passage control fibroblasts (n = 3) were cultured in the presence of medium obtained from control or IPF cell cultures for 48 h. Panel A shows a representative western blot of α-SMA (upper) and tubulin (lower) proteins and relative statistics of three independent experiments. Representative immune-blots for TGF-β1 in cell extracts and culture media of early passage control and IPF fibroblasts are shown in panel B. Culture media were concentrated by means of centrifugal filter devices YM-10 (Centricon, Millipore). In the middle, 5 ng of rhTGF-β1 has been loaded as positive control. Panel C shows real time PCR expression levels of TGF-β mRNA in control (n = 4) and IPF fibroblasts (n = 7) at early (full bars) and late (dashed bars) culture passages. Early passage IPF fibroblasts (n = 3) were treated for 30 min with a specific TGF-β receptor ALK5 inhibitor (10 µM) and changes of α-SMA expression were assessed at the protein level. Representative immune-blots for α-SMA/tubulin expression and relative statistics are reported in panel D. All data are expressed as mean fold change ± SD.
These data indicate that early passage IPF cells differ from control fibroblasts as: 1. are differentiated into myofibroblasts; 2. have a pro-fibrotic phenotype; and 3. proliferate less. Such a phenotype is not dependent upon the expression of growth factors, and especially of TGF-β, acting in an autocrine manner. Both cell types markedly slow down growth over time. This occurs along with myofibroblast differentiation with no evidence of ongoing senescence.
Oxidative Stress Induces and Maintains the Phenotype of IPF Fibroblasts
To investigate whether the phenotype of IPF cells was linked to oxidative stress we measured the expression of α-SMA and type-I collagen in control fibroblasts upon long-term challenge with H2O2. As shown in Figure 3A, α-SMA expression was robustly induced by H2O2 in a dose-dependent manner at 48 h. Time-course curves performed with a fixed concentration of H2O2 (200 µM) showed that induction of α-SMA expression increased over time, reaching the peak between 24 and 48 h (Figure 3B). Exposure to oxidative stress also accounted for higher expression of type-I collagen (Figure 3C). Under these conditions cell death was negligible, ranging from 8 to 13%. Conversely, as shown in Figure 3D, no further increase of α-SMA expression was appreciated in H2O2-treated early passage IPF fibroblasts. Short term incubation (15 min) with increasing concentrations of H2O2 induced death of early passage control fibroblasts in a dose-dependent manner (Figure 4A). Similarly, control cells death also occurred in response to a fixed concentration of H2O2 in a time-dependent manner (Figure 4B). Conversely, early passage IPF cells were resistant to peroxide-induced cell death in both culture conditions (Figure 4, panels A–B). IPF cells also retained their resistance to oxidative-induced cell death at later passages, as shown in Figure 4 (C–D). Finally, to test whether the IPF phenotype was sensitive to anti-oxidants, control and IPF fibroblasts were treated for 48 h with the ROS scavenger NAC (5 mM). Figure 5 (A–B) shows that levels of α-SMA were not modified in control fibroblasts while significantly decreased upon treatment in IPF cells at early passages. This finding was associated with a concomitant reduction of type-I collagen expression in IPF fibroblasts (Figure 5C). Conversely, no changes in α-SMA and collagen expression occurred in NAC-exposed IPF cells at late passages (Figure 5, panels D–E).
Changes of α-SMA expression were investigated by means of western blot in early control fibroblasts (n = 3) upon exogenous administration of hydrogen peroxide. Panel A shows changes of α-SMA expression upon cell treatment with increasing concentrations of H2O2 for 48 h. Conversely, panel B shows changes of α-SMA by treating cells with a fixed concentration of H2O2 (200 µM) at different time points. A representative immune-blot of α-SMA/tubulin is also shown. Panel C shows type-I collagen expression, assessed by real time PCR, in cells incubated for 48 h in the presence of H2O2 (200 µM). Expression of α-SMA in H2O2 (200 µM)-treated early passage IPF fibroblasts (n = 3) over time is shown in panel D. All data are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.001 versus basal; **p<0.05 versus basal.
Induction of cell death upon exogenous oxidative stress was assessed by means of flow cytometry in early and late passage control (n = 3) and IPF (n = 3) cells. Panels A and C show the distribution of the mean percentages of PI-positive cells treated for 15 min with increasing concentration of H2O2, in early and late passage cells, respectively. Panels B and D show the distribution of the mean percentages of PI-positive cells treated with a fixed H2O2 concentration (1 mM) at different time points in early and late passage cells, respectively. All data are expressed as mean percentage ± SD of fluorescent cells and are representative of three independent experiments. *p<0.05 versus control cells.
Control and IPF fibroblasts were treated for 48 h with the ROS scavenger NAC (5 mM). Panels A and B show representative immune-blots for α-SMA/tubulin and relative statistics in early passage control (n = 3) and IPF (n = 3) cells, respectively. Panel C illustrates changes of type-I collagen expression in early passage IPF fibroblasts. Changes of α-SMA and type-I collagen expression in NAC-treated late passage IPF fibroblasts (n = 3) are shown in panels D and E, respectively. Data are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.05 versus basal.
These data indicate that: 1. ROS stimulate normal fibroblasts to differentiate into type-I collagen expressing myofibroblasts; 2. conversely, ROS scavenging inhibits α-SMA and type-I collagen expression in IPF cells; and 3. myofibroblast differentiation of IPF cells confers resistance to oxidative stress-induced cell death.
IPF Fibroblasts Are Characterized by High ROS Levels
To dissect the nature of the signal(s) that characterizes the IPF phenotype, we measured the spontaneous ability of IPF and control fibroblasts to generate ROS. Mean basal ROS levels were significantly higher in early passage IPF fibroblasts compared with control cells, while no significant differences were recorded at late culture passages (Figure 6A). Flow cytometry analysis showed that early passage IPF fibroblasts were distributed as a cluster of highly fluorescent ROS-expressing cells (Figure 6B). Conversely, the distribution of fluorescent control cells analysed at the same culture passages was diffuse and heterogeneous. With time in culture the distribution pattern of ROS-expressing IPF cells became diffuse in a wider range of fluorescence similarly to control fibroblasts. To confirm the higher ability of IPF fibroblasts to generate ROS we measured the levels of superoxide anion released. Early passage IPF fibroblasts expressed higher superoxide anion levels compared with control cells (Figure 6C). No differences in superoxide anion release were found at late passages.
The baseline ability of control (n = 4) and IPF (n = 7) cells to generate ROS was measured at different culture passages with different methods. The intracellular content of ROS was first measured fluorimetrically in DCHF-DA loaded cells at early passages. Panel A shows levels of DCF fluorescence in early (full bars) and late (dashed bars) passage control and IPF cells. *p<0.05 versus control cells. Representative flow cytometry dot plots of the distribution of DCF fluorescence in control and IPF fibroblasts at different culture passages are shown in panel B. Panel C shows O2- production, estimated by means of superoxide dismutase-inhibitable cytocrome c reduction, respectively in early (full bars) and late (dashed bars) passage control and IPF fibroblasts. All data are reported as mean value ± SD and are representative of three independent experiments. *p<0.01 versus early passage control cells; **p<0.05 versus early passage control cells; ***p<0.001 versus early passage IPF cells.
These data indicate that early passage IPF fibroblasts: 1. express high levels of ROS and superoxide anion in comparison with control cells; and 2. are undistinguishable from control cells with respect to ROS generation at late passages.
ROS Generation in IPF Cells Occurs Through the Activation of a Membrane NADPH Oxidase-Like System
To strengthen these observations we sought to identify the source of ROS. It is known that ROS activate MEK and ERK1/2 through the inhibition of tyrosine phosphatases [22], [23]: thus, ERK phosphorylation is inducible by ROS and ROS-induced p-ERK levels are sensitive to treatment with anti-oxidants or oxidase inhibitors. Basal levels of p-ERK were analysed in control and IPF fibroblasts at different culture passages. Then, since NADPH oxidase is the main source of O2- in most cell types [24], changes of p-ERK expression were also analysed in response to cell treatment with the membrane NADPH oxidase inhibitor, DPI. As shown in Figure 7A, basal p-ERK expression was significantly higher in early passage IPF cells in comparison with the control ones. In addition, p-ERK levels significantly decreased in IPF fibroblasts in response to DPI. Conversely, control cells were insensitive to oxidase inhibition. No differences of p-ERK expression were detectable in control and IPF cells at late passages (Figure 7B). Similarly to control fibroblasts, IPF cells also became insensitive to DPI treatment. To further strengthen these observations, modulation of p-ERK levels upon H2O2 exposure was assessed in early passage control fibroblasts. As shown in Figure 7C, ERK phosphorylation was inducible in a dose dependent manner by H2O2 and this effect was inhibited by pre-treating cells with the ROS scavenger, NAC. Conversely, no further increase of p-ERK levels was detectable in response to H2O2 in early passage IPF cells that however were sensitive to NAC (Figure 7D). Finally, to address whether α-SMA expression was dependent upon ERK activation, modulation of peroxide-induced α-SMA expression was analysed in control fibroblasts pre-treated with the MEK kinase inhibitor UO126 (10 µM). As shown in Figure 7E, α-SMA was induced in response to H2O2 while was significantly decreased in UO126 pre-treated cells. Similarly, α-SMA expression did also decrease in IPF cells upon UO126 treatment.
Basal levels of ERK phosphorylation were assessed in control (n = 4) and IPF (n = 7) fibroblasts at different culture passages. Further, to determine whether a functional NADPH oxidase complex was involved in the generation of ROS, changes of p-ERK expression were analysed in response to treatment with DPI (20 µM), a flovoprotein inhibitor. Modulation of p-ERK levels was analysed upon 30 min of cell treatment. Representative immune-blots of p-ERK/tubulin and relative statistics are respectively shown in panels A and B. *p<0.001 versus basal control cells; **p<0.001 versus basal IPF cells. To investigate whether ERK signalling was inducible upon oxidative stress, early passage control (n = 3) and IPF fibroblasts (n = 3) were treated with increasing concentrations of H2O2 for 30 min. Pre-treatment of cells with the ROS scavenger NAC (10 mM for 1 h) was also analysed. Representative immune-blots and relative statistics of three independent experiments are reported in panels C (*p<0.01 versus basal; **p<0.01 versus 1 mM H2O2 treated cells) and D (*p<0.05 versus basal; **p<0.01 versus basal). To address whether ERK activation is necessary for α-SMA expression, modulation of peroxide-induced α-SMA (H2O2 200 µM for 2 h) was analysed in early passage control cells (n = 3) pre-treated with UO126 (10 mM for 30 min). Similarly, α-SMA expression was evaluated in early passage IPF fibroblasts (n = 3) in response to UO126. Panel E shows representative immune-blots of α-SMA/tubulin and relative statistics. All data are expressed as mean fold change ± SD. *p<0.05 versus H2O2 treated cells; **p<0.05 versus basal.
These data indicate that IPF fibroblasts: 1. display an active NADPH-oxidase like system accounting for ROS formation; 2. contain active ERK1/2 which is an exquisite sensor of the activation status of such a system, and 3. α-SMA expression is maintained by ROS generation through ERK activation.
Tyrosine Kinase(s) Signalling is Constitutively Activated in IPF Fibroblasts
NADPH oxidase may be activated by a wide array of receptors for growth factors, including receptor tyrosine kinase(s) (RTKs) [25]–[27]. In addition, NADPH-generated ROS can themselves sustain signalling of RTKs through the inhibition of tyrosine phosphatases [22]. To test this issue, we asked whether ROS rise in IPF cells was associated with activation of tyrosine kinase(s) signalling. Figure 8A shows that IPF cells displayed higher basal levels of tyrosine phosphorylated proteins than control cells. Levels of tyrosine phosphorylation were decreased in IPF cells at late passages (Figure 8A). Tyrosine phosphorylation was inducible by H2O2 in control fibroblasts and this effect was inhibited by pre-treatment of cells with the ROS scavenger NAC (Figure 8B). In a similar manner, p-tyrosine levels also decreased in NAC treated IPF fibroblasts (Figure 8B). These findings are relevant as corroborate previous data indicating that switch-off of RTKs may have a beneficial effect in IPF through the inhibition of myofibroblast differentiation [28]–[30]. To address whether this was the case in our setting, we measured α-SMA expression in IPF cells upon inhibition of RTKs. Although we did not analyse the specific receptor involved, inhibition of RTKs was associated with a marked reduction of α-SMA levels in IPF cells. Conversely, α-SMA expression remained unchanged in control fibroblasts (Figure 8C). However, peroxide-induced α-SMA expression was sensitive to RTKs inhibition in control fibroblasts as shown in Figure 8D.
Baseline levels of p-tyrosine proteins were assessed by western blotting in early and late passage control (n = 4) and IPF (n = 7) cells. Representative immune-blots are shown in panel A. Then, to address whether p-tyrosine signalling was inducible upon exogenous oxidative stress early passage control fibroblasts (n = 3) were treated for 15 min with increasing concentration of H2O2. Pre-treatment of cells with the ROS scavenger NAC (10 mM for 1 h) was also analysed. Modulation of p-tyrosine levels are shown in a representative immune-blot in panel B (left). Conversely, representative changes of p-tyrosine levels in early passage IPF cells (n = 3) treated with NAC (10 mM for 1 h) are shown on the right. To investigate whether myofibroblast differentiation is dependent upon tyrosine kinase signalling, changes of α-SMA expression were assessed by means of western blotting in early passage control (n = 3) and IPF (n = 5) fibroblasts treated for 30 min with the RTKs inhibitors AG1296 (anti-PDGF-R, 2 µM) and AG1478 (anti-EGF-R, 2 µM). Representative immune-blots of α-SMA/tubulin and relative statistics are shown in panel C. *p<0.001 versus untreated IPF; **p<0.01 versus untreated IPF. Finally, to investigate whether RTKs inhibition abrogates peroxide-induced α-SMA expression early passage control cells (n = 3) were pre-treated for 30 min with the RTKs inhibitors AG1296 (2 µM) and AG1478 (2 µM) and then exposed for 2 h to H2O2 (200 µM). Panel D shows representative immune-blots of α-SMA/tubulin and relative statistics. Results are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.05 versus basal; **p<0.001 versus H2O2 treated cells.
In conclusion, these observations suggest that tyrosine kinase(s) signalling: 1. is constitutively activated in IPF cells; 2. is inducible upon oxidative stress while, conversely, is inhibited by anti-oxidants; and 3. is required for myofibroblast differentiation.
Fibroblasts and myofibroblasts are key effectors in wound healing and are thought to be crucial players in IPF pathogenesis through an unabated continuation of the repair process after the resolution of inflammation [8]. ROS mediate multiple cellular functions, including proliferation, apoptosis, migration and differentiation [31], [32], all crucial events to tissue homeostasis and remodelling. Although ROS have been implicated in IPF pathogenesis, there is no conclusive evidence linking ROS metabolism to the phenotype of fibroblasts derived from IPF patients. To dissect the mechanism(s) underlying the disease, we first characterized the baseline phenotype of primary lung fibroblasts of IPF patients. Then, we focused on the redox state of IPF fibroblasts and investigated whether IPF features were influenced by oxidative stress. We found that, unlike control fibroblasts that predominantly showed a proliferative pattern, IFP cells were differentiated into myofibroblasts and displayed a pro-fibrotic phenotype. Previous studies have reported contrasting proliferative characteristics of human "fibrotic" fibroblasts [33], [34]. In our setting, IPF cells proliferated significantly less than control fibroblasts since the earliest culture passages corresponding to the first 20–25 PD. However, along with time in culture differences were no more appreciated as proliferation of IPF and control cells drastically slowed down. The absence of visible signs of senescence (β-galactosidase) which instead was associated with an increased α-SMA expression in both cell types over time in culture suggests that the progressive growth slow down was dependent on accelerated differentiation rather than on senescence [35]. IPF cells also displayed an increased ability to express higher levels of collagen expression than control fibroblasts at early passages. It is noteworthy that acquisition of the myofibroblast phenotype is necessary for collagen expression and that this effect is persistent and irreversible even after the removal of the inducing factor [36]. However, in our setting this ability was lost along with culture passages suggesting that the effect of the triggering factor was diluted out over time thus leading to the establishment of a quiescent phenotype.
In line with previous observations [37], IPF fibroblasts were committed to generate high ROS levels, and clustering of ROS-forming cells accounted for an increased amount of oxidants per cell. Interestingly, also the ability to produce ROS was lost with time in culture, as late passage IPF fibroblasts became undistinguishable from control cells. Altogether, these findings clearly suggest that IPF fibroblasts have a definitive functional lifespan and identify a precise temporal window to appreciate the differences between IPF and control fibroblasts.
As previously reported in skin fibroblasts obtained from systemic sclerosis patients [20], ROS generation in IPF fibroblasts occurred through the activation of a NADPH-like oxidase system. Indeed, levels of active ERK, which is an exquisite sensor of intracellular ROS rise, were susceptible to oxidase inhibition [19]. It has been recently shown that NADPH oxidase generation of H2O2 is required for TGF-β mediated fibrogenesis in a murine model of lung fibrosis and similar data have also been confirmed in human fibroblasts in vitro [38], [39]. In agreement with these observations we found that exogenous hydrogen peroxide strongly induced control fibroblasts to differentiate into type-I collagen expressing myofibroblasts. In addition, exposure to oxidative stress also accounted for activation of tyrosine kinase(s) signalling suggesting the existence of a link between such a pathway and cell differentiation. It is likely that the NADPH-related ROS rise in IPF cells derives from the constitutive activation of a wide array of receptors for growth factors, also including RTKs [25]–[27]. Although we did not look at the activation status of a specific tyrosine kinase receptor, we found that tyrosine kinase(s) signalling was activated in IPF cells and that RTKs activity was necessary for the maintenance of the IPF phenotype. There is evidence that in the presence of high ROS levels [19], [40] several receptors may be stabilized allowing cells to become more sensitive to sub-threshold concentrations of growth factors and cytokines. This sets on a circuitry linking growth factor receptors, including RTKs, NADPH oxidase and ROS, which self-maintains and propagates locally the inflammatory focus [19]. Far from being multiple discrete lesions, recent evidence suggests FF as a complex non-malignant reactive process responsive to local environmental stimuli. The formation of a fibroblast reticulum is consistent with a "wave" of fibrosis starting from the pleura and progressing through the lung parenchyma [41]. In this context, we suggest that the auto-amplificatory loop (RTKs, NADPH, ROS-pERK1/2) may be operating in the fibroblast reticulum where the progression of a "wave of oxidation" promotes through the freely ROS diffusion both cell to cell communication and myofibroblast differentiation.
In conclusion, the present study highlights the existence of a baseline distinctive molecular IPF phenotype characterized by an imbalanced redox state which self maintains in culture apart from the involvement of any triggering growth factor either exogenously added or acting in an autocrine manner. Although recent evidences suggest a role of autocrine TGF-β generation in maintaining the myofibroblastic phenotype in areas of fibrosis [42], we did not confirm these data in our setting. This phenotype confers resistance to oxidative stress-induced cell death, can be induced by ROS in control fibroblasts and is inhibited by ROS scavenging. Most importantly, the IPF trait is transient, since it is progressively lost along with time in culture. We suggest this measurable phenotype of IPF as an assay amenable for investigating the molecular signal(s) that initiate the events we described and for testing and validation of target specific therapies. It remains to be seen the site and the nature of the primary IPF trigger.
The authors wish to thank Dr. Adriana Gallo (Consiglio Nazionale delle Ricerche, Napoli) and Dr. Antonio Porcellini (Dipartimento di Scienze per la Salute, Università del Molise) for helpful comments.
Author Contributions
Conceived and designed the experiments: MB SA DG CV AG EVA. Performed the experiments: MB SA EF SS. Analyzed the data: MB SA DG CV AG AS EVA. Wrote the paper: MB SA EVA.
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I've posted about 40 photos from a 2010 family visit to Mesa Verde National Park in southwest Colorado, which you can reach by clicking on the link below. This was a wonderful travel experience for our family.
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Mesa Verde National Park - a Photo Album on Flickr
I've posted about 40 photos from a 2010 family visit to Mesa Verde National Park in southwest Colorado, which you can reach by clicking on the link below. This was a wonderful travel experience for our family.
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Thank you for sharing.
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Great photos and thanks for posting the link.
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Great photos. Very motivating.
How long did you spend in the Park?
Any advice on what to do to get the most out of the visit?
We're tossing up whether to spend two nights in the park or one.
The one night involves driving from Santa Fe to the Park arriving about 2 in the afternoon, buying tickets for the tour the next day, checking in and may be taking a drive. The following day doing two guided walks and a drive before heading back to Durango. Is that feasible? If we do two nights, then we would need to reduce our stay in the Durango, Silverton, Ouray area from 4 nights to 3 nights.
All suggestions welcome.
Also any suggestions on the Durango, Silverton, Ouray, Telluride area, including accommodation ideas.
Thanks in advance.
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Thank you for posting. I am a bit scared of the ladder situation so not sure I'll ever make it.
Hovenweep is another beautiful set of ruins in the area, for those interested
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Glad you enjoyed the photos. Let me try and respond to your questions:
We arrived in Mesa Verde late one afternoon, coming from Durango. We got in too late to do any sightseeing that day.
The next morning, we got up and did the following:
- Far View House (self-guided touring)
- Cliff Palace (Ranger tour)
- Balcony House (Ranger tour) - smaller cliff dwelling, but very distinctive; reached by climbing a 30' ladder and then exiting through about a 3' by 18" tunnel
- Spruce Tree House (self-guided)
- Wetherill Mesa/Long House (Ranger tour)
The next day we got up, and drove from Mesa Verde, to Four Corners, to Chaco Canyon, to Albuquerque.
I felt like we were able to cover Mesa Verde's high points. These can be done in a full day, if you plan carefully and keep moving. A key thing to keep in mind is that the approach road from the valley below Mesa Verde to the Visitor's Center/Far View Lodge takes a minimum of 45 minutes -- it's very curvy and steep.
If you get there early afternoon on one day, and leave 24 hours later, one night at Mesa Verde might suffice, but I was really glad we were there for two. Once the Park closes, you share this entire massive expanse only with the relative handful of other guests at the Far View Lodge. Our cell phones wouldn't work there. It's an amazing experience.
And by all means stay at Far View Lodge within the Park, not in Cortez.
We stayed in Durango for only one night: arrived late afternoon, took a pleasant walk along the pedestrian trails along the river, played mini-Golf and had dinner, then did the Durango-Silverton railroad trip the next day, which was great. However, the lunchstop in Silverton struck me as just about enough to see this relatively small town. I have a few photos from Dyurango, Silverton and the train trip up in another album on Flickr:
I've also done a photo album for Monument Valley:
and for Chaco Canyon:
and for the sights of Santa Fe:
and for the High Road to Taos:
and for Bandelier National Monument, NW of Santa Fe:
Can't recall where we stayed in Durango. It was a perfectly satisfactory but colorless motel on the roads leading out of town to the north. It's main advantage was its proximity to the hiking/biking trail along the river.
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Thanks, mlgb. Hovenweep is still on my list of things to see in the Four Corners area. That's a really nice set of photos. I look forward to checking out some of the others on your web page as well.
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The leader of a small Florida church that espouses anti-Islam philosophy says he is canceling plans to burn copies of the Quran on Sept. 11.
Pastor Terry Jones announced Thursday at a chaotic news conference that he decided to cancel his protest, claiming that he struck a deal with the leader of a planned Islamic Center near Ground Zero who agreed to move its controversial location.
However, Imam Rauf, the leader of the proposed center did not make a deal and has never talked to Pastor Terry Jones, Park51 officials tell Huffington Post, adding that they are open to all solutions and that the proposed Islamic center is open to dialogue and reconciliation with the community in lower Manhattan.
Jones's plans to burn Islam's holiest text Saturday sparked an international outcry.
President Barack Obama, the top U.S. general in Afghanistan and several Christian leaders had urged Jones to reconsider his plans. They said his actions would endanger U.S. soldiers and provide a strong recruitment tool for Islamic extremists. Jones' protest also drew criticism from religious and political leaders from across the Muslim world.
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iFanboy.com - The Place To Discuss Comics, By Fans for Fans. iFanboy.com reviews the best comic books released each week.
March 20th, 2016
Episode 729 of 797 episodes
This week, Conor Kilpatrick just woke up, Ron Richards does his best to wrestle with hotel wi-fi, and Josh Flanagan takes on a new and surprising title: Defender of Continuity. Running Time: 00:56:10 Comics: 00:01:30 – Superman: American Alien #5 00:09:54 - Power Man and Iron Fist #2 00:13:21 - International Iron Man #1 00:17:21 - The Infinity Entity #2 00:20:22 - Spider-Woman #5 00:26:19 - Starve #7 00:28:23 - Superman #50 00:31:54 - Huck #5 00:33:11 - All-New X-Men #6/Extraordinary X-Men #8 Audience Questions: 00:37:56 – Greg B. from Oakland, CA likes Invincible but hates Invincible. 00:43:26 - Patrick F. from Greenville, NC wants to take a look at Stan Lee's legacy. Brought To You By: Amazon - Get all your comics, DVDs, video games, electronics and more and help iFanboy out in the process! iFanboy Members - Become one today for $3/month or $30/year! Or make a one time donation of any amount! Music: "I Need Never Get Old" Nathaniel Rateliff and The Night Sweats
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