Posts Tagged ‘Cancer – General’

Nathalie’s Story: A Health Journey With A Happy Ending

Patient was diagnosed with adenocarcinoma of the duodenum over two years ago and had tumor removed at age 35. Interview was conducted 2+ years post-surgery.

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures


Nathalie Monette of Laval, the third largest city in Quebec, Canada, counts her blessings each and every day. The 35-year-old is looking forward to making her mark on a bright and promising future as a newly married woman with a supportive family, new job as head of internal communications for a public service organization, and a new lease on life. Diagnosed a little over two years ago with a rare cancer called adenocarcinoma of the duodenum, Nathalie never envisioned that her life would take many twists and turns before she and her doctors arrived at an optimal treatment regimen.

Nathalie describes some of the classic warning signs she had for about six months before her actual medical diagnosis: abdominal cramping, nausea, vomiting, acid reflux and loss of weight.

“I felt sick all the time. I was losing weight and had pain in my upper abdomen after eating. My condition was getting worse with each week. My boyfriend, Jeff, at the time, who is now my husband, took me to several doctors who initially listened to my list of symptoms, examined me and told me to take antacids and avoid stress – and sent me home. It was increasingly becoming more difficult to manage my life, my relationships, and my job.”

The doctors in one hospital that she visited even considered she might be having a cardiovascular incident, since she was vomiting, was nauseous and had a stomach ache. Her blood levels were normal, which didn’t help the doctors, who, again, could find no serious health issue and sent her home.

Image SOURCE: Photographs courtesy of Nathalie Monette on the day of her wedding to Jeff. Top Left: Nathalie with her parents, Céline and Jean-Claude. Top Right: Nathalie with Jeff, and her two sisters, Julie and Marie-Claude. Below Right: Nathalie and Jeff.

For the next few weeks, Nathalie visited hospital after hospital in search of finding a more steadfast diagnosis of her condition – and a doctor who would listen to her and treat her symptoms.

“I was weak and vomiting. At this point, I kept losing weight — about 40 pounds in a total of six months.”

She decided to take the situation in her own hands and changed her diet, eliminating gluten, spices, and other major food groups. Nothing seemed to relieve her symptoms. She knew reading about possible medical conditions on the internet could cause additional stress. Having worked in the pharmaceutical industry, she was glad she knew where to look and what sources of information could be trusted.

Continued Search For Answers

“The medical system in Quebec is complicated,” she said. “In this public system, there is no family doctor assigned to you who follows your care year after year. And since I was perceived by the system as a young, relatively healthy woman, I was put on a waiting list for 3 to 4 years to be assigned to a general practitioner.”

Frustrated, hopeless and fearful for her health, Jeff got more involved in her diagnosis and took her to yet another hospital. Nathalie’s search took her from hospital to hospital and doctor to doctor with no known diagnosis.

“I was very angry, disappointed and at the end my rope. I just wanted to feel better and live my life.”

Then, one day, there was a ray of hope – and it took six months to find it. At a nearby hospital called Hôpital de St-Eustache where Jeff decided to take her, she came across two young physicians, Dr. Annie-Claude Bergeron, an emergency room doctor, and Dr. Marie-Hélène Gingras, a gastroenterologist, who happened to be Nathalie’s same age. Dr. Bergeron listened to her symptoms, examined her, and was determined to help her. A day later, Dr. Gingras ran several diagnostic tests, including an endoscopy and ultrasound, and more specialized blood tests.

“While undergoing the endoscopy, the doctor couldn’t find anything remarkable and was about to remove it. She decided to push the camera 5cm farther into my duodenum – and found the cause of my illness.”

Finally, Nathalie had definitive results. She had a 3½ cm (1.4 inches) tumor in her duodenum.

Dr. Gingras was devastated by the news she had to share. She called specialists in Montreal who would operate on Nathalie. Dr. Simon Turcotte, physician, hepatopancreatobiliary and liver transplantation expert who specializes in gastrointestinal cancer immunobiology and solid tumor immunotherapy, took her case.

“When Dr. Gingras told me about my condition, I was relieved and afraid at the same time. My heart sank when I got the news.”

Nathalie had a rare cancerous condition that only shows up in a handful of older people. It also was unusual that the tumor was situated in the duodenum rather than the colon, where most tumors of this variety normally occur. She also didn’t have history of that type of cancer in her family. She couldn’t even be tested for any genetic markers, since no genes have been identified as markers for this rare condition.

So, three weeks later, Nathalie was transferred to Hôpital Saint-Luc in Montreal, for a, hopefully, life-saving surgery. She had to trust her new expert, Dr. Turcotte, with her life.

“There was no room for error in removing the tumor. It was situated 1mm from my pancreas and every other vital organ I needed to survive.”

By nature, Nathalie is a strong, fiercely independent woman and there was no doubt she would come through the operation with flying colors.

About one month after surgery, she was scheduled for six months of chemotherapy to ensure that the cancer was eradicated. One day every two weeks, she received a powerful cocktail of Folfox (Leucovorin®, 5-FU, Adrucil® and Eloxatin®).

“Because of the chemotherapy, I had a minimal appetite, could not taste any food, could not drink or touch anything cold and needed to keep my weight at the same level.”

Her parents, Céline and Jean-Claude, two sisters, Julie and Marie-Claude, and Jeff, of course — were of great support and encouragement for her. Jeff insisted to meet with her nutritionist to determine a health plan so that she received the necessary nutrients in her food. Because Nathalie could not taste any food because of the chemotherapy, she tricked her mind by eating meals that she remembered from her childhood days. In that way, she was transported back in time mentally and she thought about the great food she had when she was growing up. Her parents were always on hand to cook these traditional meals that were filled with protein, spices, salt and fat to give her the added boost (and some taste) to help her system recover.

Duodenum, A Complex, Powerful Organ

Nathalie describes the duodenum as a complex organ – a C-shaped, hollow tube about 25-38 cm (10-15 inches) long, largely responsible for the enzymatic breakdown of food in the small intestine.

“This small but powerful organ is the shortest part of the small intestine which regulates the rate of how the stomach empties.”

According to the Inner Body web site, the duodenum receives partially digested food, called chyme, from the stomach and plays a vital role in the chemical digestion of chyme in preparation for absorption in the small intestine. Many chemical secretions from the pancreas, liver and gallbladder mix with the chyme in the duodenum to help chemical digestion.

Back to Normal

Nathalie’s life is back to normal, as much as it can be after such a medical ordeal.

“The past is just the past. I try not to think about the trauma that I’ve been through. I look forward as that is what is important.”

She got married last August (2015) to Jeff, who demonstrated his love to her the best way possible in caring for her throughout this ordeal. They met on the internet in 2010, at a moment when Nathalie wanted to leave the dating scene to focus on personal projects. They talked, met shortly after, and became great friends. Only a year later did Nathalie accept to be in a relationship with Jeff.

“About one week after my surgery when I was home, Jeff proposed marriage to me. I was visiting my family for Easter and Jeff had prepared everything. He had first asked my parents for my hand in marriage in the hospital a few weeks prior to my surgery. Then he prepared a charade with answers that related to the strength of his feelings for me. Funny enough, I did not understand what was going on at that point. Little did I know, he was declaring his love and it’s when he showed me a ring that I understood. Of course, I was overwhelmed with emotion and very touched that he got my family involved in the event.

“I am under regular care of my medical team of seven doctors – a gastroenterologist, oncologist surgeon, family general practitioner and many other specialists. I’ve had follow-up appointments at three months, six months, and one year. Those appointments include a gastroscopy, colonoscopy, scan, and blood tests, and so far, my health is the best ever. I like to tease the doctors when I see my charts – I look like an athlete on paper! In our Canadian medical system, each specialist treats only that part of the body. I make sure that all my test results are xeroxed and sent in advance of my appointment to each doctor. That takes time, but I am assured that everyone sees the same test results and can make educated decisions. That also makes for a more holistic view of my life.”

Advocate for Patient’ Rights

“Knowledge, access to information and caregiver support are probably the three most important factors in patient care. Medicine on its own is just not enough. Patients need a support system to balance out the highs and lows of searching through a medical condition, diagnosis and treatment plan. I hope one day to advocate for patient voices as it is a much needed part of our medical system.

“In hindsight, I realize all the doctors who saw me during the six months that I suffered prior to my diagnosis could not have known about my condition, unless they ran more tests. Surprisingly, I had done blood tests before that time for long-term disability insurance. The insurer had refused to insure me without explanation. Starting to be very sick, I did not pursue the work with them to understand their decision. Unfortunately, I learned a few weeks after my surgery that their test revealed the count of a certain type of protein was too high, therefore, too risky for them to insure me. They knew I was seriously sick but took about eight months to let me know. Had the insurer shared their results sooner, had doctors ran similar blood tests, or done a scan, I would have been diagnosed way sooner, which could have resulted in not needing chemotherapy.”

Incidence of Adenocarcinoma

Adenocarcinomas or malignant tumors of the duodenum are extremely rare, uncommon and difficult to manage and treat, according to Drs. P.L. Fagniez and N. Rotman in a book chapter in Surgical Treatment – Evidence-Based and Problem-Oriented, a medical textbook that assesses currently accepted clinical practice that takes into account when recommendations for patient treatment are made.The tumors represent 0.3 percent of gastrointestinal tract tumors and up to 50 percent of small bowel malignancies. They may arise from duodenal polyps or they may be associated with Celiac Disease. Five-year-survival varies widely according to published reports in the medical literature, but it is generally reported to be greater than 40 percent if the tumor is surgically removed.

Due to the low incidence of the disease globally, there is no randomized study comparing different types of treatment. In fact, the medical literature only discusses a small number of patients with this condition, who are usually older, or patients who are seen over a period of time. The treatment plan is complete surgical removal of the tumor, which is the only hope for a cure. Nonetheless, good long-term results have been observed with segmented tumor removal, particularly for tumors of the distal part of the duodenum, according to the same book chapter mentioned in the paragraph above.

A Bright Future Ahead

Nathalie believes in second chances and the value of waking up each and every day to new challenges and opportunities.

“Life is to be lived and enjoyed. I love what I do and I cherish my relationships, my work and my free time. In whatever I do, I give 100 percent.”

She believes she is very lucky to have had the diagnosis at this time of her life.

“In a way, my parents, my family, my husband were always present in my health journey. They followed up on doctors’ appointments, helped me with daily living chores, researched the medical literature, contacted new doctors, and generally, were my sounding board on everything. They were invaluable to me and it was my privilege that I am blessed with such a supportive family.

“I believe the road is set for you in life and it is up to all of us to seize the moment. My condition has given me strength to explore who I am and validate the way I always approach life.”

Nathalie Monette provided her permission to publish this interview on July 30, 2016.


Search Title:

Duodenum AND Cancer | Open Studies | Exclude Unknown in Database. The search was conducted on July 30, 2016 and there were  45 studies found.

Presented, below, is a Subset of Clinical Trials on the List of 45 Studies related to Duodenum AND Cancer

SEE LINK, Below for the list of clinical trials currently recruiting:

Subset of Clinical Trials on the List of 45 Studies – Duodenum AND Cancer (6)

Or you may click on the following individual links below for clinical trials that are currently recruiting:

Spectroscopy From Duodenum

Condition: Pancreatic Adenocarcinoma
Intervention: Other: Spectroscopy device

A Randomized Trial of Two Surgical Techniques for Pancreaticojejunostomy in Patients Undergoing Pancreaticoduodenectomy

Conditions: Pancreatic Neoplasms;   Biliary Tract Neoplasms;   Pancreatitis, Chronic;   Duodenal Neoplasms
Intervention: Procedure: pancreaticojejunostomy

Endoscopic Characteristics of Duodenal and Ampullary Lesions

Condition: Duodenal Diseases
Intervention: Other: Tissue Sampling

EUS GUIDED Transduodenal Biopsy Using the 19G Flex

Condition: Abdominal Neoplasms
Intervention: Device: Expect™19Flex needle (Boston Scientific Corp.,Natick,MA,USA)

Study of Gastroduodenal Metallic Stent vs Gastrojejunostomy

Condition: Gastric Cancer
Interventions: Device: gastroduodenal stent placement;   Procedure: gastrojejunostomy

Prevalence of Small Bowel Polyps in Patients With Sporadic Duodenal Adenomas

Condition: Polyps
Intervention: Device: Small bowel video capsule endoscopy (VCE) GIVEN/COVIDIEN LTD

Long-term Outcomes of Endoscopic Resection (ER) of Lesions of the Duodenum and Ampulla

Condition: Adenoma, Villous
Intervention: Procedure: Endoscopic Mucosal Resection

Prophylactic Octreotide to Prevent Post Duodenal EMR and Ampullectomy Bleeding

Condition: Adenoma
Interventions: Drug: octreotide;   Other: No octreotide


The Use of a Restrictive Fluid Regimen With Hypertonic Saline for Patients Undergoing Pancreaticoduodenectomy

Condition: Pancreaticoduodenectomy
Interventions: Drug: 3% NaCl Solution;   Drug: Lactated Ringers Solution

Effects of Pancreaticoduodenectomy on Glucose Metabolism

Conditions: Diabetes Mellitus;   Glucose Intolerance
Intervention:  —




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Gastrointestinal Endocrinology





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Marcela’s Story:  A Liver Transplant Gives the Gift of Life

Patient is HCV Positive, liver transplanted from a 22-year-old donor performed at age 70. Interview conducted 14 years post-liver transplant.

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures

For Marcela Almada Calles of Valle de Bravo, Mexico, a picturesque town on the shores of Lake Avándaro about two hours outside of Mexico City where she has lived for 30 years, life is about seizing the moment and having “an open mind and positive attitude.”  An active woman in her 80’s, Marcela’s days are full of professional and personal achievements and a long list of activities still to accomplish. However, life wasn’t always so positive as she put her life on hold for two-and-a-half years to relocate to Los Angeles, California, so that she could have a liver transplant.

“My spirit and attitude have always been what has carried me through life and difficult situations. This time was no different.”

Image SOURCE: Photographs courtesy of Marcela Almada Calles.   

Marcela’s story started 20 years ago during a time when she operated a successful event planning and catering business for high-profile government and social dignitaries, pharmaceutical companies, and luxury department stores.

“I normally worked long hours from early morning until evening, until one day, I felt exceptionally tired and it became a huge effort to concentrate. My ankles were swollen and I was out of breath all the time and my skin was yellow. I felt sleepy and would sometimes become tired during the day. This was unusual for me. I knew something was not right.”

At that point, Marcela decided to make an appointment with her local physician and friend, Dr. Sergio Ulloa, a highly regarded rheumatologist and corporate and government affairs leader in Mexico, who examined her and took several blood tests. When the blood results came back, Dr. Ulloa immediately referred her to Dr. Sergio Kershenovich, a well-regarded hepatologist, at his private clinic, who checked her for symptoms of Hepatitis C. After that Marcela decided to get another opinion and went to see Dr. Fernando Quijano, a general surgeon, who immediately wanted her to have surgery because he had found a cancerous tumor in her liver.

“My doctors’ opinions were that I needed to have a liver transplant immediately because I was in liver failure. It appeared that I had a failing liver — and a tumor there as well and my liver was not working properly.”

Relocating Life to the United States

At that point, my six children – Marcela, Luis, Diego, Rodolfo, Gabriela, Mario — who live in parts of Mexico and Singapore became involved in my health care decisions and treatment plan.

“My son, Luis, believed the best treatment for me was to see a liver specialist in the United States so that I received the best care from a leading liver transplantation hospital. He made some connections with friends and that next day, Dr. Francisco Durazo, chief of Transplant Hepatology and medical director of the Dumont UCLA Liver Transplant Center in Los Angeles, told me to come immediately to see him. I remember my children were supportive and concerned, but were afraid for me as we all knew that I had a long road ahead of me.”

At that time, she was put on a national liver transplant list by the UCLA Transplant Center.

“What I didn’t know was that more than 9,000 potential recipients are currently awaiting liver transplants.”

“Dr. Durazo was very concerned and told me that my liver was not working at all and I had to have a liver transplant as soon as possible, so he asked me to stay in Los Angeles, since I was now part of a transplant list.”

Evaluation By Transplant Team

Marcela’s case is no different than any other patient awaiting a liver transplant. According to their web site, the UCLA Transplant Center conducts evaluations over two or three days. During this time, the patients meets with a social worker, transplant hepatologist, surgeon, transplant coordinator, psychiatrist and dietitian, as well as other specialists as needed. The evaluation is customized to each patient’s medical condition. Once the evaluation is completed, each patient’s case is presented at a weekly meeting of the UCLA Liver Transplant Consultation Team. This group includes specialists from surgery, adult and pediatric hepatology, cardiology, pulmonary, nephrology, hematology, infectious disease, as well as transplant coordinators and social workers. At this time, the team determines if any other tests are required to ensure the patient’s candidacy for transplant, then the patient and the physician are notified of the recommendation made by the transplant team.

Waiting For Answers

Marcela arrived at UCLA in Los Angeles with her family on Mother’s Day — May 10, 1999 — for what she describes as “the best time in her life to be alive with the help of medicine and technology.” That meant that she needed to rent an apartment and live near the hospital in case the doctors received an anonymous donor who would give her the gift of life.

“I had to wear a beeper 24 hours a day and I was never alone. My children took turns over the next two-and-a-half years to give up their lives with their families to live with me and help me navigate the health care system and my upcoming surgery.”

Marcela filled her days at her new apartment in Los Angeles reading about her condition, meditating to quiet her mind, watching television, and talking with family, friends and neighbors.

“The doctors called me two times over the next few months, saying they had an anonymous liver donor and I needed to come now to the hospital for tests. Unfortunately, those blood tests and other diagnostic tests showed that I was not a good match, so the doctors sent me home. It was a frustrating time because I wanted to have the liver transplant surgery and move on with my life.”

Finally, after waiting eight months for a liver transplant, Marcela’s outlook on life was greatly improved when an anonymous donor gave her the gift of life – a new, healthy liver.

“The donor’s blood type was a match for me. The surgery took eight hours and it was successful. The doctors told me that my immune system might reject my new liver, so I was given a cocktail of medicines, such as anti-rejection drugs, corticosteroids, calcinurin inhibitors, mTOR inhibitors, and antibiotics and watched very closely in the hospital.”

Marcela was then permitted to leave the hospital only a week after her surgery.

“That was the happiest day of my life. My spirits were high and I had a life to live.”

Her children served as her strength.

“My children took turns flying back and forth to Los Angeles to stay with me. They had a long list of instructions from the doctor. I could take some walks and eat small meals for the next few weeks, but I couldn’t exert myself in any way. I developed a cold over the next few weeks, as my immune system was low, so I had to take special care to eat right, get enough sleep and, most of all, relax. My body, spirit and mind had much healing to do.”

For the next 1 ½ years, Los Angeles was my “second” home.

“I needed to remain there after the procedure so my doctors could monitor my progress. During that time, I felt stronger each day. The support of my family was a true blessing for me. They were my eyes and ears – and my greatest advocates. My doctor recommended that I come weekly for check-ups and go through a physical therapy program so that I could regain my liver function and physical strength. I followed all my doctor’s orders.”

Day by day, Marcela believed as if she could conquer the world.

“I decided, one day many months after the surgery, to become ‘irresponsible’ and spent time with a few good friends, Gabriela and Guadalupe, who traveled to see me. For a weekend, we went to Las Vegas to see shows and go to the casinos. I laughed, played and walked all I could. My children didn’t even know what I was up to, but I felt good and wanted to enjoy the world and my new freedom.”

Marcela was able to return home to Valle de Bravo with a fresh perspective, a long list of things to do, and many happy memories.

“Since that time, I have kept myself active and busy; I never let my mind and heart rest. I am also forever grateful to my anonymous liver donor because it is because of a 22-year-old young man who died in an unfortunate automobile accident that I am here today.”

Liver Transplant Facts

The liver is the body’s vital organ that you cannot live without. It serves many critical functions, including metabolism of drugs and toxins, removing degradation products of normal body metabolism and synthesis of many proteins and enzyme, which are necessary for blood to clot. Transplantation is the only cure for liver insufficiency or liver failure because no device or machine reliably performs all the functions of the liver.–procedures/liver-transplantation.aspx

According to a hospital transplant web site, overall, outcomes for liver transplantation are very good, but vary significantly depending on the indication for liver transplant as well as factors associated with the donor. Currently, the overall patient survival one year after liver transplant is 88 percent. Patient survival five years after liver transplant is 73 percent. These results vary significantly based on the indication for liver transplantation. The encouraging trend is that over the past 20 years short- and long-term patient survival has continued to improve. With advances in surgical technique, organ preservation, peri-operative care, and immunosuppression, survival will hopefully continue to improve in the future.–procedures/liver-transplantation.aspx

Life For Marcela Today

Science is helping rebalance medicine with the most innovative discoveries and new ways of treating illness.

“I am happy to be part of the solution with a happy ending, too.”

Today, Marcela leads a rich and full life.

“It’s been 14 years since my liver transplant. I continue to feel healthy and alive. Nothing will keep me from doing what I want to do.”

Marcela has an active social life. She takes frequent vacations around the world, including a three-month holiday to Asia, where she travels multiple times to Bali, Cambodia, China and Singapore, where her daughter lives. She is an avid golfer and organizes tournaments in many private golf courses. She is learning to speak French, which is an easy transition (she says) from speaking Spanish. She plays cards with a group of friends weekly, sings in a musical group, and takes dance lessons, too. Life is very, very good.

Editor’s note: We would like to thank Gabriela Contreras, a global communications consultant and patient advocate, for the tremendous help and support that she provided in locating and scheduling time to talk with Marcela Almada Calles.

Marcela Almada Calles provided her permission to publish this interview on July 21, 2016.



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Retrieved from–procedures/liver-transplantation.aspx

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Technologies For Targeting And Delivering Chemotherapeutics Directly To The Tumour Site


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Personalized Immunotherapy: The Immuno-Oncology Summit August 30-31 2016 Boston MA

Reporter: Stephen J Williams, PhD




Leaders in Pharmaceutical Business intelligence (LPBI) Group will cover in Real Time using Social Media

The CHI’S 4TH ANNUAL IMMUNO-ONCOLOGY SUMMIT – Personalized Immunotherapy

Personalized Oncology in the Genomic Era: Expanding the Druggable Space

Aviva Lev-Ari, PhD, RN

will be streaming LIVE from the Marriott Long Wharf Hotel in Boston, MA





Plenary Keynotes


Matthew Goldstein

4:00 A New Era of Personalized Therapy: Using Tumor Neoantigens to Unlock the Immune System

Matthew J. Goldstein, M.D., Ph.D., Director, Translational Medicine, Neon Therapeutics, Inc.

Neon Therapeutics, Inc. launched in 2015 to focus on advancing neoantigen biology to improve cancer patient care. A neoantigen-based product engine will allow Neon to develop further treatment modalities including next-generation vaccines and T cell therapies targeting both personalized as well as shared neoantigens. The company’s first trial will launch later this year investigating the combination of a personalized, vaccine with nivolumab in advanced Melanoma, NSCLC, and Bladder Cancer.

Michael Rosenzweig

4:30 Emerging Innate Immune Targets for Enhancing Adaptive Anti-Tumor Responses

Michael Rosenzweig, Ph.D., Executive Director, Biology-Discovery, IMR Early Discovery, Merck Research Laboratories

Novel cancer immunotherapies targeting T cell checkpoint proteins have emerged as powerful tools to induce profound, durable regression and remission of many types of cancer. Despite these advances, multiple studies have demonstrated that not all patients respond to these therapies, and the ability to predict which patients may respond is limited. Harnessing the innate immune system to augment the adaptive anti-tumor response represents an attractive target for therapy, which has the potential to enhance both the percentage and rate of response to checkpoint blockade.


Morganna Freeman

5:00 Reading Tea Leaves:
The Dilemma of Prediction and Prognosis in Immunotherapy

Morganna Freeman, D.O., Associate Director, Melanoma & Cutaneous Oncology Program, The Angeles Clinic and Research Institute

With the rapid expansion of immunotherapeutics in oncology, scientifically significant advances have been made with both the depth and duration of antitumor responses. However, not all patients benefit, or quickly relapse, thus much scientific inquiry has been devoted to appropriate patient selection and how such obstacles might be overcome. While more is known about potential biomarkers, accurate prognostication persists as a knowledge gap, and efforts to bridge it will be discussed here.

Personalized Immunotherapy | The Immuno-Oncology Summit
August 30-31, 2016 | Marriott Long Wharf Hotel – Boston, MA

Personalized Immunotherapy
Personalized Oncology in the Genomic Era: Expanding the Druggable Space
August 30-31, 2016 | Learn More | Sponsorship & Exhibit Opportunities | Register by July 29 & SAVE up to $200!

Fueled with advances in genomic technologies, personalized oncology promises to innovate cancer therapy and target the previously undruggable space. Developments in immune checkpoint inhibitors, cancer vaccines, and adoptive T-cell therapies, as well as biomarker-driven immuno-oncology clinical trials, are enabling the next generation of cancer therapy. Cambridge Healthtech Institute’s Inaugural Personalized Immunotherapy meeting brings together clinical immuno-oncologists and thought leaders from pharmaceutical and biotech companies, and leading academic teams to share research and case studies in implementing patient-centric approaches to using the immune system to beat cancer.


Basics of Personalized Immunotherapy: What Is a Good Antigen?
Pramod K. Srivastava, M.D., Ph.D., Professor, Immunology and Medicine, Director, Carole and Ray Neag Comprehensive Cancer Center, University of Connecticut School of Medicine

Novel Antibodies against Immunogenic Neoantigens
Philip M. Arlen, M.D., President & CEO, Precision Biologics, Inc.

PD-1 Blockade in Tumors with Mismatch-Repair Deficiency
Luis Alberto Diaz, M.D., Associate Professor, Oncology, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center


Cancer Vaccines in the Era of Checkpoint Inhibitors
Keith L. Knutson, Ph.D., Professor, Immunology, Mayo Clinic

Developing Therapeutic Cancer Vaccine Strategies for Prostate Cancer
Ravi Madan, M.D., Clinical Director, Genitourinary Malignancies Branch, National Cancer Institute, National Institutes of Health

Getting Very Personal: Fully Individualized Tumor Neoantigen-Based Vaccine Approaches to Cancer Therapy
Karin Jooss, Ph.D., CSO, Gritstone Oncology

Approaches to Assess Tumor Mutation Load for Selecting Patients for Cancer Immunotherapy
John Simmons, Ph.D., Manager, Research Services, Personal Genome Diagnostics

In situ Vaccination for Lymphoma
Joshua Brody, M.D., Director, Lymphoma Immunotherapy Program, Icahn School of Medicine at Mount Sinai

Immunotherapy Using Ad5 [E1-, E2b-] Vector Vaccines in the Cancer MoonShot 2020 Program
Frank R. Jones, Ph.D., Chairman & CEO, Etubics Corporation


Integration of Natural Killer-Based Therapy into the Treatment of Lymphoma
Andrew M. Evens, D.O., Professor and Chief, Hematology/Oncology, Tufts University School of Medicine; Director, Tufts Cancer Center

Dendritic Cells: Personalized Cancer Vaccines and Inducers of Multi-Epitope-Specific T Cells for Adoptive Cell Therapy
Pawel Kalinski, M.D., Ph.D., Professor, Surgery, Immunology, and Bioengineering, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute

Mesothelin-Targeted CAR T-Cell Therapy for Solid Tumors
Prasad S. Adusumilli, M.D., FACS, Deputy Chief of Translational & Clinical Research, Thoracic Surgery, Memorial Sloan-Kettering Cancer Center

Synthetic Regulation of T Cell Therapies Adds Safety and Enhanced Efficacy to Previously Unpredicted Therapies
David M. Spencer, Ph.D., CSO, Bellicum Pharmaceuticals

Long-Term Relapse-Free Survival of Patients with Acute Myeloid Leukemia (AML) Receiving a Telomerase- Engineered Dendritic Cell Immunotherapy
Jane Lebkowski, Ph.D., President & CSO, Research and Development, Asterias Biotherapeutics

Activated and Exhausted Tumor Infiltrating B Cells in Non-Small Cell Lung Cancer Patients Present Antigen and Influence the Phenotype of CD4 Tumor Infiltrating T Cells
Tullia Bruno, Ph.D., Research Assistant Professor, Immunology, University of Pittsburgh

About the Immuno-Oncology Summit

CHI’s 4th Annual Immuno-Oncology Summit has been designed to support a coordinated effort by industry players to bring commercial immunotherapies and immunotherapy combinations through clinical development and into the market. This weeklong, nine-meeting set will include topics ranging from early discovery through clinical development as well as emerging areas such as oncolytic virotherapy. Overall, this event will provide a focused look at how researchers are applying new science and technology in the development of the next generation of effective and safe immunotherapies.

Monday, August 29 –
Tuesday, August 30
Tuesday, August 30 –
Wednesday, August 31
Thursday, September 1 –
Friday, September 2
Immunomodulatory Antibodies Combination Immunotherapy Adoptive T Cell Therapy
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GE Healthcare has acquired Biosafe Group SA, a supplier of Integrated Cell Bioprocessing Systems for Cell Therapy and Regenerative Medicine Industry

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Researchers of University of Texas at San Antonio, USA, have developed a new, non-invasive method which can kill cancer cells in two hours, an advance that may significantly help people with inoperable or hard-to-reach tumours, as well as young children stricken with the deadly disease.


The method involves injecting a chemical compound, nitrobenzaldehyde, into the tumour and allowing it to diffuse into the tissue. A beam of light is then aimed at the tissue, causing the cells to become very acidic inside and, essentially, commit suicide. Within two hours, up to 95 per cent of the targeted cancer cells are estimated to be dead.


The method was tested against triple negative breast cancer, one of the most aggressive types of cancer and one of the hardest to treat. The prognosis for triple negative breast cancer is usually very poor. One treatment in the laboratory was able to stop the tumour from growing and doubled the chances of survival in the mice.


According to the researchers all forms of cancer attempt to make cells acidic on the outside and attract the attention of blood vessels as an attempt to get rid of the acid. But, instead, the cancer cells latches onto the blood vessel and uses it to make the tumour grow bigger.


Chemotherapy treatments target all cells in the body, and certain chemotherapeutics try to keep cancer cells acidic as a way to kill the cancer. This is what causes many cancer patients to lose their hair and become weak. This method however, is more precise and can target just the tumour.


This research is presently extended on drug-resistant cancer cells to make this therapy as strong as possible. The researchers also started to develop a nanoparticle that can be injected into the body to target metastasised cancer cells. The nanoparticle is activated with a wavelength of light which can pass harmlessly through skin, flesh and bone and still activate the nanoparticle.


This non-invasive method will help cancer patients with tumours in areas that have proven problematic for surgeons, such as the brain stem, aorta or spine. It could also help people who have received the maximum amount of radiation treatment and can no longer cope with the scarring and pain that goes along with it, or children who are at risk of developing mutations from radiation as they grow older.




Nuha Buchanan Kadri, Matthew Gdovin, Nizar Alyassin, Justin Avila, Aryana Cruz, Louis Cruz, Steve Holliday, Zachary Jordan, Cameron Ruiz and Jennifer Watts. Photodynamic acidification therapy to reduce triple negative breast cancer growth in vivo. Journal of Clinical Oncology, Vol 34, No 15_suppl (May 20 Supplement), 2016: e12574.


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Vectorisation Of Immune Checkpoint Inhibitor Antibodies

Reporter: David Orchard-Webb, PhD


The FDA approved ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) combination in October 2015 for the treatment of advanced melanoma. The antibodies have recently been approved in the UK for the same indication. Over half of patients respond to the combination [1]. These drugs belong to the class of monoclonal antibodies known as immune checkpoint inhibitors. The binding of anti-CTLA-4 antibodies to activated T cells prevents the surface CTLA-4 receptor from binding CD80 and/or CD86 on antigen presenting cells (APCs). Normally CTLA-4 binding to APCs deactivates the T-cell. Antibodies against programmed cell death protein 1 (PD-1) work by a similar mechanism to CTLA-4. These drugs are delivered by repeated intravenous injections (iv) [2].


Oncolytic viruses are an emerging class of immunotherapeutics that actively stimulate the immune system by releasing tumour antigens via lysis and by virtue of anti-viral immunity. The first FDA approved oncolytic virus (Imlygic), developed by Amgen/ BioVex, was given the green light in October 2015 for advanced melanoma patients delivered via direct tumour injection. The mechanism of action of oncolytic viruses is highly complementary with checkpoint inhibitor antibodies and multiple trials combining these two classes of agent are under way.


At the recent American Association for Cancer Research (AACR) annual meeting in New Orleans, Louisiana, the oldest biotechnology company in France – Transgene, presented preclinical data concerning oncolytic vaccinia viruses that express whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv) against mouse PD-1 [3]. These combinations proved superior over virus alone in mouse xenografts of melanoma and fibrosarcoma cell lines. Transgene claim that “these results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs” (Figure 1) [3].


Figure 1: The convergence of therapeutics based on oncolytic viruses and monoclonal antibodies against immune checkpoint inhibotor proteins. Image Source: Eric Molina. No changes were made. Creative Commons Attribution 2.0 Generic (CC BY 2.0).


The combination of immune checkpoint inhibitors and oncolytic virus as a single molecular entity clearly has advantages in terms of manufacturing cost effectiveness. In addition viral vectors have the capacity for perfect specificity to tumours which has potential safety advantages.






Other Related Articles Published In This Open Access Online Journal Include The Following:

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Insights into the Metabolome

Curator: Larry H. Bernstein, MD, FCAP



Updated 6/3/2016


Tapping the Metabolome

Genes, Transcripts, Proteins—All Have Come into Their “-Ome”     GEN May 15, 2016 (Vol. 36, No. 10)



The retina is responsible for capturing images from the visual field. Retinitis pigmentosa, which refers to a group of inherited diseases that cause retinal degeneration, causes a gradual decline in vision because retinal photoreceptor cells (rods and cones) die. Images on the left are courtesy of the National Eye Institute, NIH; image on the right is courtesy of Robert Fariss, Ph.D., and Ann Milam, Ph.D., National Eye Institute, NIH.

Metabolomics, the comprehensive evaluation of the products of cellular processes, can provide new findings and insight in a vast array of diseases and dysfunctions. Though promising, metabolomics lacks the standing of genomics or proteomics. It is, in a manner of speaking, the new kid on the “omics” block.

Even though metabolomics is still an emerging discipline, at least some quarters are giving it a warm welcome. For example, metabolomics is being advanced by the Common Fund, an initiate of the National Institutes of Health (NIH). The Common Fund has established six national metabolomics cores. In addition, individual agencies within NIH, such as the National Institute of Environmental Health Sciences (NIEHS), are releasing solicitations focused on growing more detailed metabolomics programs.

Whether metabolomic studies are undertaken with or without public support, they share certain characteristics and challenges. Untargeted or broad-spectrum studies are used for hypotheses generation, whereas targeted studies probe specific compounds or pathways. Reproducibility is a major challenge in the field; many studies cannot be reproduced in larger cohorts. Carefully defined guidance and standard operating procedures for sample collection and processing are needed.

While these challenges are being addressed, researchers are patiently amassing metabolomic insights in several areas, such as retinal diseases, neurodegenerative diseases, and autoimmune diseases. In addition, metabolomic sleuths are availing themselves of a growing selection of investigative tools.

A Metabolomic Eye on Retinal Degeneration

The retina has one of the highest metabolic activities of any tissue in the body and is composed of multiple cell types. This fact suggests that metabolomics might be helpful in understanding retinal degeneration. At least, that’s what occurred to Ellen Weiss, Ph.D., a professor of cell biology and physiology at the University of North Carolina School of Medicine at Chapel Hill. To explore this possibility, Dr. Weiss began collaborating with Susan Sumner, Ph.D., director of systems and translational sciences at RTI International.

Retinal degeneration is often studied through the use of genetic-mouse models that mimic the disease in humans. In the model used by Dr. Weiss, cells with a disease-causing mutation are the major light-sensing cells that degenerate during the disease. Individuals with the same or a similar genetic mutation will initially lose dim-light vision then, ultimately, bright-light vision and color vision.

Wild-type and mutant phenotypes, as well as dark- and light-raised animals, were compared, since retinal degeneration is exacerbated by light in this genetic model. Retinas were collected as early as day 18, prior to symptomatic disease, and analyzed. Although data analysis is ongoing, distinct differences have emerged between the phenotypes as well as between dark- and light-raised animals.

“There is a clear increase in oxidative stress in both light-raised groups but to a larger extent in the mutant phenotype,” reports Dr. Weiss. “There are global changes in metabolites that suggest mitochondrial dysfunction, and dramatic changes in lipid profiles. Now we need to understand how these metabolites are involved in this eye disease and the relevance of these perturbations.”

For example, the glial cells in the retina that upregulate a number of proteins in response to stress to attempt to save the retina are as likely as the light-receptive neurons to undergo metabolic changes.

“One of the challenges in metabolomics studies is assigning the signals that represent the metabolites or compounds in the samples,” notes Dr. Sumner. “Signals may be ‘unknown unknowns,’ compounds that have never been identified before, or ‘known unknowns,’ compounds that are known but that have not yet been assigned in the biological matrix.”

Internal and external libraries, such as the Human Metabolome Dictionary, are used to match signals. Whether or not a match exists, fragmentation patterns are used to characterize the metabolite, and when possible a standard is obtained to confirm identity. To assist with this process, the NIH Common Fund supports Metabolite Standard Synthesis Cores (MSSCs). RTI International holds an MSSC contract in addition to being a NIH-designated metabolomics core.

Mitochondrial Dysfunction in Alzheimer’s Disease     

Alzheimer’s disease (AD) is difficult to diagnose early due to its asymptomatic phase; accurate diagnosis occurs only in postmortem brain tissue. To evaluate familial AD, a rare inherited form of the disease, the laboratory of Eugenia Trushina, Ph.D., associate professor of neurology and associate professor of pharmacology at the Mayo Clinic, uses mouse models to study the disease’s early molecular mechanisms.

Synaptic loss underlies cognitive dysfunction. The length of neurons dictates that mitochondria move within the cell to provide energy at the site of the synapses. An initial finding was that very early on mitochondrial trafficking was affected reducing energy supply to synapses and distant parts of the cell.

During energy production, the major mitochondrial metabolite is ATP, but the organelle also produces many other metabolites, molecules that are implicated in many pathways. One can assume that changes in energy utilization, production, and delivery are associated with some disturbance.

“Our goal,” explains Dr. Trushina, “was to get a proof of concept that we could detect in the blood of AD patients early changes of mitochondria dysfunction or other changes that could be informative of the disease over time.”

A Mayo Clinic aging study involves a cohort of patients, from healthy to those with mild cognitive impairment (MCI) through AD. Patients undergo an annual battery of tests including cognitive function along with blood and cerebrospinal fluid sampling. Metabolic signatures in plasma and cerebrospinal fluid of normal versus various disease stages were compared, and affected mitochondrial and lipid pathways identified in MCI patients that progressed to AD.

“Last year we published on a new compound that goes through the blood/brain barrier, gets into mitochondria, and very specifically, partially inhibits mitochondrial complex I activity, making the cell resistant to oxidative damage,” details Dr. Trushina. “The compound was able to either prevent or slow the disease in the animal familial models.

“Treatment not only reduced levels of amyloid plaques and phosphorylated tau, it also restored mitochondrial transport in neurons. Now we have additional compounds undergoing investigation for safety in humans, and target selectivity and engagement.”

“Mitochondria play a huge role in every aspect of our lives,” Dr. Trushina continues. “The discovery seems counterintuitive, but if mitochondria function is at the heart of AD, it may provide insight into the major sporadic form of the disease.”

Distinguishing Types of Asthma

In children, asthma generally manifests as allergy-induced asthma, or allergic asthma. And allergic asthma has commonalities with allergic dermatitis/eczema, food allergies, and allergic rhinitis. In adults, asthma is more heterogeneous, and distinct and varied subpopulations emerge. Some have nonallergic asthma; some have adult-onset asthma; and some have obesity-, occupational-, or exercise-induced asthma.

Adult asthmatics may have markers of TH2 high verus TH2 low asthma (T helper 2 cell cytokines) and they may respond to various triggers—environmental antigens, occupational antigens, irritants such as perfumes and chlorine, and seasonal allergens. Exercise, too, can trigger asthma.

One measure that can phenotype asthmatics is nitric oxide, an exhaled breath biomarker. Nitric oxide is a smooth muscle relaxant, vasodilator, and bronchodilator that can have anti-inflammatory properties. There is a wide range of values in asthmatics, and a number of values are needed to understand the trend in a particular patient. L-arginine is the amino acid that produces nitric oxide when converted to L-citrulline, a nonessential amino acid.

According to Nicholas Kenyon, M.D., a pulmonary and critical care specialist who is co-director of the University of California, Davis Asthma Network (UCAN), some metabolomic studies suggest that there is a state of L-arginine depletion during asthma attacks or in severe asthma suggesting a lack of substrate to produce nitric oxide. Dr. Kenyon is conducting clinical work on L-arginine supplementation in a double-blind cross-over  intervention trial of L-arginine versus placebo. The 50-subject study in severe asthmatics should be concluded in early 2017.

Many new biologic therapies are coming to market to treat asthma; it will be challenging to determine which advanced therapy to provide to which patient. Therapeutics mostly target severe asthma populations and are for patients with evidence of higher numbers of eosinophils in the blood and lung, which include anti-IL-5 and (soon) anti-IL-13, among others.

Tools Development 

Waters is developing metabolomics applications that use multivariate statistical methods to highlight compounds of interest. Typically these applications combine separation procedures, accomplished by means of liquid chromatography or gas chromatography (LC or GC), with detection methods that rely on mass spectrometry (MS). To support the identification, quantification, and analysis of LC-MS data, the company provides bioinformatics software. For example, Progenesis QI software can interrogate publicly available databases and process information about isotopic patterns, retention times, and collision cross-sections.

Mass spectrometry (MS) is the gold standard in metabolomics and lipidomics. But there is a limit to what accurate mass and resolution can achieve. For example, neither isobaric nor isomeric species are resolvable solely by MS. New orthogonal analytical tools will allow more confident identifications.

To improve metabolomics separations before MS detection, a post-ionization separation tool, like ion mobility, which is currently used to support traditional UPLC-MS and MS imaging metabolomics protocols, becomes useful. The collision-cross section (CCS), which measures the shape of molecules, can be derived, and it can be used as an additional identification coordinate.

Other new chromatographic tools are under development, such as microflow devices and UltraPerformance Convergence Chromatography (UPC2), which uses liquid CO2 as its mobile phase, to enable new ways of separating chiral metabolites. Both UPC2 and microflow technologies have decreased solvent consumption and waste disposal while maintaining UPLC-quality performance in terms of chromatographic resolution, robustness, and reproducibility.

Informatics tools are also improving. In the latest versions of Waters’ Progenesis software, typical metabolomics identification problems are resolved by allowing interrogation of publicly available databases and scoring according to accurate mass, isotopic pattern, retention time, CCS, and either theoretical or experimental fragments.

MS imaging techniques, such as MALDI and DESI, provide spatial information about the metabolite composition in tissues. These approaches can be used to support and confirm traditional analyses without sample extraction, and they allow image generation without the use of antibodies, similar to immunohistochemistry.

“Ion-mobility tools will soon be implemented for routine use, and the use of extended CCS databases will help with metabolite identification,” comments Giuseppe Astarita Ph.D., principal scientist, Waters. “More applications of ambient ionization MS will emerge, and they will allow direct-sampling analyses at atmospheric pressure with little or no sample preparation, generating real-time molecular fingerprints that can be used to discriminate among phenotypes.”

Microflow Technology   

Microflow technology offers sensitivity and robustness. For example, at the Proteomics and Metabolomics Facility, Colorado State University, peptide analysis was typically performed using nanoflow chromatography; however, nanoflow chromatography is slow and technically challenging. Moving to microflow offered significant improvements in robustness and ease-of-use and resulted in improved chromatography without sacrificing sensitivity.

Conversely, small molecule applications were typically performed with analytical-scale chromatography. While this flow regime is extremely robust and fast, it can sometimes be limited in sensitivity. Moving to microflow offered significant improvements in sensitivity, 5- to 10-fold depending on the compound, without sacrificing robustness.

But broad-scale microflow adoption is hampered by a lack of available column chemistries and legacy HPLC or UPLC infrastructure that is not conducive to low-flow operation.

“We utilize microflow technology on all of our tandem quadrupole instruments for targeted quantitative assays,” says Jessica Prenni, Ph.D., director, Proteomics and Metabolomics Facility, Colorado State University. “All of our peptide quantitation is exclusively performed with microflow technology, and many of our small molecule assays. Application examples include endocannabinoids, bile acids and plant phytohormone panels.”

Compound annotation and comparability and transparency in data processing and reporting is a challenge in metabolomics research. Multiple groups are actively working on developing new tools and strategies; common best practices need to be adopted.

The continued growth of open-source spectral databases and new tools for spectral prediction from compound databases will dramatically impact the ability for metabolomics to result in novel discoveries. The move to a systems-level understanding through the combination of various omics data also will have a huge influence and be enabled by the continued development of open-source and user-friendly pathway-analysis tools.

 Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging

Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.

In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.

In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.

At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.

Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.

Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.

Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.

Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.

“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”

In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.

Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.

“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”

Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.

“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”

This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.

Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).

Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response.  …..


Researchers Uncover How ‘Silent’ Genetic Changes Drive Cancer

Fri, 06/03/2016 – 8:41amby Rockeller University

“Traditionally, it has been hard to use standard methods to quantify the amount of tRNA in the cell,” says Tavazoie. The lead authors of the article, Hani Goodarzi, formerly a postdoc in the lab and now a new assistant professor at UCSF, and research assistant Hoang Nguyen, devised and applied a new method that utilizes state-of-the-art genomic sequencing technology to measure the amount of tRNAs in different cell types.

The team chose to compare breast tissue from healthy individuals with tumor samples taken from breast cancer patients–including both primary tumors that had not spread from the breast to other body sites, and highly aggressive, metastatic tumors.

They found that the levels of two specific tRNAs were significantly higher in metastatic cells and metastatic tumors than in primary tumors that did not metastasize or healthy samples. “There are four different ways to encode for the protein building block arginine,” explains Tavazoie. “Yet only one of those–the tRNA that recognizes the codon CGG–was associated with increased metastasis.”

The tRNA that recognizes the codon GAA and encodes for a building block known as glutamic acid was also elevated in metastatic samples.

The team hypothesized that the elevated levels of these tRNAs may in fact drive metastasis. Working in mouse models of primary, non-metastatic tumors, the researchers increased the production of the tRNAs, and found that these cells became much more invasive and metastatic.

They also did the inverse experiment, with the anticipated results: reducing the levels of these tRNAs in metastatic cells decreased the incidence of metastases in the animals.

How do two tRNAs drive metastasis? The researchers teamed up with members of the Rockefeller University proteomics facility to see how protein expression changes in cells with elevated levels of these two tRNAs.

“We found global increases in many dozens of genes,” says Tavazoie, “so we analyzed their sequences and found that the majority of them had significantly increased numbers of these two specific codons.”

According to the researchers, two genes stood out among the list. Known as EXOSC2 and GRIPAP1, these genes were strongly and directly induced by elevated levels of the specific glutamic acid tRNA.

“When we mutated the GAA codons to GAG– a “silent” mutation because they both spell out the protein building block glutamic acid–we found that increasing the amount of tRNA no longer increased protein levels,” explains Tavazoie. These proteins were found to drive breast cancer metastasis.

The work challenges previous assumptions about how tRNAs function and suggests that tRNAs can modulate gene expression, according to the researchers. Tavazoie points out that “it is remarkable that within a single cell type, synonymous changes in genetic sequence can dramatically affect the levels of specific proteins, their transcripts, and the way a cell behaves.”


Testing Blood Metabolites Could Help Tailor Cancer Treatment

6/03/2016 1 Comment by Institute of Cancer Research

Scientists have found that measuring how cancer treatment affects the levels of metabolites – the building blocks of fats and proteins – can be used to assess whether the drug is hitting its intended target.

This new way of monitoring cancer therapy could speed up the development of new targeted drugs – which exploit specific genetic weaknesses in cancer cells – and help in tailoring treatment for patients.

Scientists at The Institute of Cancer Research, London, measured the levels of 180 blood markers in 41 patients with advanced cancers in a phase I clinical trial conducted with The Royal Marsden NHS Foundation Trust.

They found that investigating the mix of metabolic markers could accurately assess how cancers were responding to the targeted drug pictilisib.

Their study was funded by the Wellcome Trust, Cancer Research UK and the pharmaceutical company Roche, and is published in the journal Molecular Cancer Therapeutics.

Pictilisib is designed to specifically target a molecular pathway in cancer cells, called PI3 kinase, which has key a role in cell metabolism and is defective in a range of cancer types.

As cancers with PI3K defects grow, they can cause a decrease in the levels of metabolites in the bloodstream.

The new study is the first to show that blood metabolites are testable indicators of whether or not a new cancer treatment is hitting the correct target, both in preclinical mouse models and also in a trial of patients.

Using a sensitive technique called mass spectrometry, scientists at The Institute of Cancer Research (ICR) initially analysed the metabolite levels in the blood of mice with cancers that had defects in the PI3K pathway.

They found that the blood levels of 26 different metabolites, which were low prior to therapy, had risen considerably following treatment with pictilisib. Their findings indicated that the drug was hitting its target, and reversing the effects of the cancer on mouse metabolites.

Similarly, in humans the ICR researchers found that almost all of the metabolites – 22 out of the initial 26 – once again rose in response to pictilisib treatment, as seen in the mice.

Blood levels of the metabolites began to increase after a single dose of pictilisib, and were seen to drop again when treatment was stopped, suggesting that the effect was directly related to the drug treatment.

Metabolites vary naturally depending on the time of day or how much food a patient has eaten. But the researchers were able to provide the first strong evidence that despite this variation metabolites can be used to test if a drug is working, and could help guide decisions about treatment.


New Metabolic Pathway Reveals Aspirin-Like Compound’s Anti-Cancer Properties

Researchers at the Gladstone Institutes say they have found a new pathway by which salicylic acid, a key compound in the nonsteroidal anti-inflammatory drugs aspirin and diflunisal, stops inflammation and cancer.

In a study (“Salicylate, Diflunisal and Their Metabolites Inhibit CBP/p300 and Exhibit Anticancer Activity”) published in eLife, the investigators discovered that both salicylic acid and diflunisal suppress two key proteins that help control gene expression throughout the body. These sister proteins, p300 and CREB-binding protein (CBP), are epigenetic regulators that control the levels of proteins that cause inflammation or are involved in cell growth.

By inhibiting p300 and CBP, salicylic acid and diflunisal block the activation of these proteins and prevent cellular damage caused by inflammation. This study provides the first concrete demonstration that both p300 and CBP can be targeted by drugs and may have important clinical implications, according to Eric Verdin, M.D., associate director of the Gladstone Institute of Virology and Immunology .

“Salicylic acid is one of the oldest drugs on the planet, dating back to the Egyptians and the Greeks, but we’re still discovering new things about it,” he said. “Uncovering this pathway of inflammation that salicylic acid acts upon opens up a host of new clinical possibilities for these drugs.”

Earlier research conducted in the laboratory of co-author Stephen D. Nimer, M.D., director of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, and a collaborator of Verdin’s, established a link between p300 and the leukemia-promoting protein AML1-ETO. In the current study, scientists at Gladstone and Sylvester worked together to test whether suppressing p300 with diflunisal would suppress leukemia growth in mice. As predicted, diflunisal stopped cancer progression and shrunk the tumors in the mouse model of leukemia. ……


Novel Protein Agent Targets Cancer and Host of Other Diseases

Researchers at Georgia State University have designed a new protein compound that can effectively target the cell surface receptor integrin v3, mutations in which have been linked to a number of diseases. Initial results using this new molecule show its potential as a therapeutic treatment for an array of illnesses, including cancer.

The novel protein molecule targets integrin v3 at a novel site that has not been targeted by other scientists. The researchers found that the molecule induces apoptosis, or programmed cell death, of cells that express integrin v3. This integrin has been a focus for drug development because abnormal expression of v3 is linked to the development and progression of various diseases.

“This integrin pair, v3, is not expressed in high levels in normal tissue,” explained senior study author Zhi-Ren Liu, Ph.D., professor in the department of biology at Georgia State. “In most cases, it’s associated with a number of different pathological conditions. Therefore, it constitutes a very good target for multiple disease treatment.”

“Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, which binds to integrin αvβ3 outside the classical ligand-binding site,” the authors wrote. “We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3.”

The findings from this study were published recently in Nature Communications in an article entitled “Rational Design of a Protein That Binds Integrin αvβ3 Outside the Ligand Binding Site.”   …..

“We took a unique angle,” Dr. Lui noted. “We designed a protein that binds to a different site. Once the protein binds to the site, it directly triggers cell death. When we’re able to kill pathological cells, then we’re able to kill the disease.”

The investigators performed extensive cell and molecular testing that confirmed ProAgio interacts and binds well with integrin v3. Interestingly, they found that ProAgio induces apoptosis by recruiting caspase 8—an enzyme that plays an essential role in programmed cell death—to the cytoplasmic area of integrin v3. ProAgio was much more effective in inducing cell death than other agents tested.


Noncoding RNAs Not So Noncoding

Bits of the transcriptome once believed to function as RNA molecules are in fact translated into small proteins.

By Ruth Williams | June 1, 2016

In 2002, a group of plant researchers studying legumes at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, discovered that a 679-nucleotide RNA believed to function in a noncoding capacity was in fact a protein-coding messenger RNA (mRNA).1 It had been classified as a long (or large) noncoding RNA (lncRNA) by virtue of being more than 200 nucleotides in length. The RNA, transcribed from a gene called early nodulin 40 (ENOD40), contained short open reading frames (ORFs)—putative protein-coding sequences bookended by start and stop codons—but the ORFs were so short that they had previously been overlooked. When the Cologne collaborators examined the RNA more closely, however, they found that two of the ORFs did indeed encode tiny peptides: one of 12 and one of 24 amino acids. Sampling the legumes confirmed that these micropeptides were made in the plant, where they interacted with a sucrose-synthesizing enzyme.

Five years later, another ORF-containing mRNA that had been posing as a lncRNA was discovered inDrosophila.2,3 After performing a screen of fly embryos to find lncRNAs, Yuji Kageyama, then of the National Institute for Basic Biology in Okazaki, Japan, suppressed each transcript’s expression. “Only one showed a clear phenotype,” says Kageyama, now at Kobe University. Because embryos missing this particular RNA lacked certain cuticle features, giving them the appearance of smooth rice grains, the researchers named the RNA “polished rice” (pri).

Turning his attention to how the RNA functioned, Kageyama thought he should first rule out the possibility that it encoded proteins. But he couldn’t. “We actually found it was a protein-coding gene,” he says. “It was an accident—we are RNA people!” The pri gene turned out to encode four tiny peptides—three of 11 amino acids and one of 32—that Kageyama and colleagues showed are important for activating a key developmental transcription factor.4

Since then, a handful of other lncRNAs have switched to the mRNA ranks after being found to harbor micropeptide-encoding short ORFs (sORFs)—those less than 300 nucleotides in length. And given the vast number of documented lncRNAs—most of which have no known function—the chance of finding others that contain micropeptide codes seems high.

Overlooked ORFs

From the late 1990s into the 21st century, as species after species had their genomes sequenced and deposited in databases, the search for novel genes and their associated mRNAs duly followed. With millions or even billions of nucleotides to sift through, researchers devised computational shortcuts to hunt for canonical gene and mRNA features, such as promoter regions, exon/intron splice sites, and, of course, ORFs.

ORFs can exist in practically any stretch of RNA sequence by chance, but many do not encode actual proteins. Because the chance that an ORF encodes a protein increases with its length, most ORF-finding algorithms had a size cut-off of 300 nucleotides—translating to 100 amino acids. This allowed researchers to “filter out garbage—that is, meaningless ORFs that exist randomly in RNAs,” says Eric Olsonof the University of Texas Southwestern Medical Center in Dallas.

Of course, by excluding all ORFs less than 300 nucleotides in length, such algorithms inevitably missed those encoding genuine small peptides. “I’m sure that the people who came up with [the cut-off] understood that this rule would have to miss anything that was shorter than 100 amino acids,” saysNicholas Ingolia of the University of California, Berkeley. “As people applied this rule more and more, they sort of lost track of that caveat.” Essentially, sORFs were thrown out with the computational trash and forgotten.

Aside from statistical practicality and human oversight, there were also technical reasons that contributed to sORFs and their encoded micropeptides being missed. Because of their small size, sORFs in model organisms such as mice, flies, and fish are less likely to be hit in random mutagenesis screens than larger ORFs, meaning their functions are less likely to be revealed. Also, many important proteins are identified based on their conservation across species, says Andrea Pauli of the Research Institute of Molecular Pathology in Vienna, but “the shorter [the ORF], the harder it gets to find and align this region to other genomes and to know that this is actually conserved.”

As for the proteins themselves, the standard practice of using electrophoresis to separate peptides by size often meant micropeptides would be lost, notes Doug Anderson, a postdoc in Olson’s lab. “A lot of times we run the smaller things off the bottom of our gels,” he says. Standard protein mass spectrometry was also problematic for identifying small peptides, says Gerben Menschaert of Ghent University in Belgium, because “there is a washout step in the protocol so that only larger proteins are retained.”

But as researchers take a deeper dive into the function of the thousands of lncRNAs believed to exist in genomes, they continue to uncover surprise micropeptides. In February 2014, for example, Pauli, then a postdoc in Alex Schier’s lab at Harvard University, discovered a hidden code in a zebrafish lncRNA. She had been hunting for lncRNAs involved in zebrafish development because “we hadn’t really anticipated that there would be any coding regions out there that had not been discovered—at least not something that is essential,” she says. But one lncRNA she identified actually encoded a 58-amino-acid micropeptide, which she called Toddler, that functioned as a signaling protein necessary for cell movements that shape the early embryo.5

Then, last year, Anderson and his colleagues reported another. Since joining Olson’s lab in 2010, Anderson had been searching for lncRNAs expressed in the heart and skeletal muscles of mouse embryos. He discovered a number of candidates, but one stood out for its high level of sequence conservation—suggesting to Anderson that it might have an important function. He was right, the RNA was important, but for a reason that neither Anderson nor Olson had considered: it was in fact an mRNA encoding a 46-amino-acid-long micropeptide.6

“When we zeroed in on the conserved region [of the gene], Doug found that it began with an ATG [start] codon and it terminated with a stop codon,” Olson says. “That’s when he looked at whether it might encode a peptide and found that indeed it did.” The researchers dubbed the peptide myoregulin, and found that it functioned as a critical calcium pump regulator for muscle relaxation.

With more and more overlooked peptides now being revealed, the big question is how many are left to be discovered. “Were there going to be dozens of [micropeptides]? Were there going to be hundreds, like there are hundreds of microRNAs?” says Ingolia. “We just didn’t know.”

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Research at Micro- and Nanoscales

From whole cells to genes, closer examination continues to surprise.

By Mary Beth Aberlin | June 1, 2016–and-Nanoscales

Little things mean a lot. To any biologist, this time-worn maxim is old news. But it’s worth revisiting. As several articles in this issue of The Scientist illustrate, how researchers define and examine the “little things” does mean a lot.

Consider this month’s cover story, “Noncoding RNAs Not So Noncoding,” by TS correspondent Ruth Williams. Combing the human genome for open reading frames (ORFs), sequences bracketed by start and stop codons, yielded a protein-coding count somewhere in the neighborhood of 24,000. That left a lot of the genome relegated to the category of junk—or, later, to the tens of thousands of mostly mysterious long noncoding RNAs (lncRNAs). But because they had only been looking for ORFs that were 300 nucleotides or longer (i.e., coding for proteins at least 100 amino acids long), genome probers missed so-called short ORFs (sORFs), which encode small peptides. “Their diminutive size may have caused these peptides to be overlooked, their sORFs to be buried in statistical noise, and their RNAs to be miscategorized, but it does not prevent them from serving important, often essential functions, as the micropeptides characterized to date demonstrate,” writes Williams.

How little things work definitely informs another field of life science research: synthetic biology. As the functions of genes and gene networks are sussed out, bioengineers are using the information to design small, synthetic gene circuits that enable them to better understand natural networks. In “Synthetic Biology Comes into Its Own,” Richard Muscat summarizes the strides made by synthetic biologists over the last 15 years and offers an optimistic view of how such networks may be put to use in the future. And to prove him right, just as we go to press, a collaborative group led by one of syn bio’s founding fathers, MIT’s James Collins, has devised a paper-based test for Zika virus exposure that relies on a freeze-dried synthetic gene circuit that changes color upon detection of RNAs in the viral genome. The results are ready in a matter of hours, not the days or weeks current testing takes, and the test can distinguish Zika from dengue virus. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” commented one researcher about the test.

Moving around little things is the name of the game when it comes to delivering a package of drugs to a specific target or to operating on minuscule individual cells. Mini-scale delivery of biocompatible drug payloads often needs some kind of boost to overcome fluid forces or size restrictions that interfere with fine-scale manipulation. To that end, ingenious solutions that motorize delivery by harnessing osmotic changes, magnets, ultrasound, and even bacterial flagella are reviewed in “Making Micromotors Biocompatible.”


Cilengitide: The First Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesis and Clinical Evaluation

Anticancer Agents Med Chem. 2010 Dec; 10(10): 753–768.
doi:  10.2174/187152010794728639

Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany).

This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.

Integrins are heterodimeric receptors that are important for cell-cell and cell-extracellular matrix (ECM) interactions and are composed of one α and one β-subunit [1, 2]. These cell adhesion molecules act as transmembrane linkers between their extracellular ligands and the cytoskeleton, and modulate various signaling pathways essential in the biological functions of most cells. Integrins play a crucial role in processes such as cell migration, differentiation, and survival during embryogenesis, angiogenesis, wound healing, immune and non-immune defense mechanisms, hemostasis and oncogenic transformation [1]. The fact that many integrins are also linked with pathological conditions has converted them into very promising therapeutic targets [3]. In particular, integrins αvβ3, αvβ5 and α5β1 are involved in angiogenesis and metastasis of solid tumors, being excellent candidates for cancer therapy [47].

There are a number of different integrin subtypes which recognize and bind to the tripeptide sequence RGD (arginine, glycine, aspartic acid), which represents the most prominent recognition motif involved in cell adhesion. For example, the pro-angiogenic αvβ3 integrin binds various RGD-containing proteins, including fibronectin (Fn), fibrinogen (Fg), vitronectin (Vn) and osteopontin [8]. It is therefore not surprising that this integrin has been targeted for cancer therapy and that RGD-containing peptides and peptidomimetics have been designed and synthesized aiming to selectively inhibit this receptor [9, 10].

One classical strategy used in drug design is based on the knowledge about the structure of the receptor-binding pocket, preferably in complex with the natural ligand. However, this strategy, the so-called “rational structure-based design”, could not be applied in the field of integrin ligands since the first structures of integrin’s extracellular head groups were not described until 2001 for αvβ3 [11] (one year later, in 2002 the structure of this integrin in complex with Cilengitide was also reported [12]) and 2004 for αIIbβ3 [13]. Therefore, initial efforts in this field focused on a “ligand-oriented design”, which concentrated on optimizing RGD peptides by means of different chemical approaches in order to establish structure-activity relationships and identify suitable ligands.

We focused our interest in finding ligands for αvβ3 and based our approach on three chemical strategies pioneered in our group: 1) Reduction of the conformational space by cyclization; 2) Spatial screening of cyclic peptides; and 3)N-Methyl scan.

The combination of these strategies lead to the discovery of the cyclic peptidec(RGDf(NMe)V) in 1995. This peptide showed subnanomolar antagonistic activity for the αvβ3 receptor, nanomolar affinities for the closely related integrins αvβ5 and α5β1, and high selectivity towards the platelet receptor αIIbβ3. The peptide was patented together with Merck in 1997 (patent application submitted in 15.9.1995, opened in 20.3.1997) [14] and first presented with Merck’s agreement at the European Peptide Symposium in Edinburgh (September 1996) [15]. The synthesis and activity of this molecule was finally published in 1999 [16]. This peptide is now developed by Merck-Serono, (Darmstadt, Germany) under the name “Cilengitide” and has recently entered Phase III clinical trials for treating glioblastoma [17].  …..

The discovery 30 years ago of the RGD motif in Fn was a major breakthrough in science. This tripeptide sequence was also identified in other ECM proteins and was soon described as the most prominent recognition motif involved in cell adhesion. Extensive research in this direction allowed the description of a number of bidirectional proteins, the integrins, which were able to recognize and bind to the RGD sequence. Integrins are key players in the biological function of most cells and therefore the inhibition of RGD-mediated integrin-ECM interactions became an attractive target for the scientific community.

However, the lack of selectivity of linear RGD peptides represented a major pitfall which precluded any clinical application of RGD-based inhibitors. The control of the molecule’s conformation by cyclization and further spatial screening overcame these limitations, showing that it is possible to obtain privileged bioactive structures, which enhance the biological activity of linear peptides and significantly improve their receptor selectivity. Steric control imposed in RGD peptides together with their biological evaluation and extensive structural studies yielded the cyclic peptide c(RGDfV), the first small selective anti-angiogenic molecule described. N-Methylation of this cyclic peptide yielded the much potentc(RGDf(NMe)V), nowadays known as Cilengitide.

The fact that brain tumors, which are highly angiogenic, are more susceptible to the treatment with integrin antagonists, and the positive synergy observed for Cilengitide in combination with radio-chemotherapy in preclinical studies, encouraged subsequent clinical trials. Cilengitide is currently in phase III for GBM patients and in phase II for other types of cancers, with to date a promising therapeutic outcome. In addition, the absence of significant toxicity and excellent tolerance of this drug allows its combination with classical therapies such as RT or cytotoxic agents. The controlled phase III study CENTRIC was launched in 2008, with primary outcome measures due on September 2012. The results of this and other clinical studies are expected with great hope and interest.

Integrin Targeted Therapeutics

Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Integrins are heterodimeric cell surface receptors found in nearly all metazoan cell types, composed of non-covalently linked α and β subunits. In mammals, eighteen α-subunits and eight β-subunits have been identified to date 1. From this pool, 24 distinct heterodimer combinations have been observed in vivo that confer cell-to-cell and cell-to-ligand specificity relevant to the host cell and the environment in which it functions 2. Integrin-mediated interactions with the extracellular matrix (ECM) are required for the attachment, cytoskeletal organization, mechanosensing, migration, proliferation, differentiation and survival of cells in the context of a multitude of biological processes including fertilization, implantation and embryonic development, immune response, bone resorption and platelet aggregation. Integrins also function in pathological processes such as inflammation, wound healing, angiogenesis, and tumor metastasis. In addition, integrin binding has been identified as a means of viral entry into cells 3. ….

Combination of cilengitide and radiation therapy and temozolomide. The addition of cilengitide to radiotherapy and temozolomide based treatment regimens has shown promising preliminary results in ongoing Phase II trials in both newly diagnosed and progressive glioblastoma multiforme 139140. In addition to the Phase II objectives sought, these trials are significant in that they represent progress that has made in determining tumor drug uptake and in identifying a subset of patients that may benefit from treatment. In a Phase II trial enrolling 52 patients with newly diagnosed glioblastoma multiforme receiving 500 mg cilengitide twice weekly during radiotherapy and in combination with temozolomide for 6 monthly cycles following radiotherapy, 69% achieved 6 months progression free survival compared to 54 % of patients receiving radiotherapy followed by temozolomide alone. The one-year overall survival was 67 and 62 % of patients for the cilengitide combination group and the radiotherapy and temozolomide group, respectively. Non-hematological grade 3-4 toxcities were limited, and included symptoms of fatigue, asthenia, anorexia, elevated liver function tests, deep vein thrombosis and pulmonary embolism in across a total of 5.7% of the patients. Grade 3-4 hematological malignancies were more common and included lymphopenia (53.8%), thrombocytopenia (13.4%) and neutropenia (9.6%). This trial is significant in the fact that is has provided the first evidence correlating a molecular biomarker with response to treatment. Decreased methylguanine methyltransferase (MGMT) expression was associated with favorable outcome. Patients harboring increased MGMT promoter methylation appeared to benefit more from combined treatment with cilengitide than did patients lacking promoter methylation. The significance of the MGMT promoter methylation in predicting response is likely due to inclusion of temozolomide in the treatment combination.

A similar Phase II study evaluating safety and differences in overall survival among newly diagnosed glioblastoma multiforme patients receiving radiation therapy combined with temozolomide and varying doses of cilengitide is nearing completion. Preliminary reports specify that initial safety run-in studies in 18 patients receiving doses 500, 1000 and 2000 mg cilengitide found no dose limiting toxicities. Subsequently 94 patients were randomized to receive standard therapy plus 500 or 2000 mg cilengitide. Median survival time in both cohorts was 18.9 months. At 12 months the overall survival was 79.5 % (89/112 patients).

In the last two decades great progress has been made in the discovery and development of integrin targeted therapeutics. Years of intense research into integrin function has provided an understanding of the potential applications for the treatment of disease. Advances in structural characterization of integrin-ligand interactions has proved beneficial in the design and development of potent, selective inhibitors for a number of integrins involved in platelet aggregation, inflammatory responses, angiongenesis, neovascularization and tumor growth.

The αIIbβ3 integrin antagonists were the first inhibitors to make their way into clinical use and have proven to be effective and safe drugs, contributing to the reduction of mortality and morbidity associated with acute coronary syndromes. Interestingly, the prolonged administration of small molecules targeting this integrin for long-term prevention of thrombosis related complications have not been successful, for reasons that are not yet fully understood. This suggests that modulating the intensity, duration and temporal aspects of integrin function may be more effective than simply shutting off integrin signaling in some instances. Further research into the dynamics of platelet activation and thrombosis formation may elucidate the mechanisms by which integrin activation is modulated.

The introduction of α4 targeted therapies held great promise for the treatment of inflammatory diseases. The development of Natalizumab greatly improved the quality of life for multiple sclerosis patients and those suffering with Crohn’s Disease compared to previous treatments, but the role in asthma related inflammation could not be validated. Unfortunately for MS and Crohn’s patients, immune surveillance in the central nervous system was also compromised as a direct effect α4β7 antagonism, with potentially lethal effects. Thus Natalizumab and related α4β7 targeting drugs are now limited to patients refractory to standard therapies. The design and development of α4β1 antagonists for the treatment of Crohn’s Disease may offer benefit with decreased risks. The involvement of these integrins in fetal development also raises concerns for widespread clinical use.

Integrin antagonists that target angiogenesis are progressing through clinical trials. Cilengitide has shown promising results for the treatment of glioblastomas and recurrent gliomas, cancers with notoriously low survival and cure rates. The greatest challenge facing the development of anti-angiogenic integrin targeted therapies is the overall lack of biomarkers by which to measure treatment efficacy.


Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach

Biochem J. 2009 Nov 11; 424(2): 179–189. doi:  10.1042/BJ20090992
Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by α5β1 is poorly understood. Here we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of RGD and synergy sites in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish α5 subunit with the corresponding regions of human α5 we show that blades 1-4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller (D3-A3 loop) contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human α5β1 supports an important function for D3-A3 loop residues Trp-157 and Ala-158 in the binding of antagonists. These results will aid the development of reagents that block α5β1 functions in vivo.
Structural Basis of Integrin Regulation and Signaling
Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cellpathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 Å couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
The immune system relies heavily on integrins for (a) adhesion during leukocyte trafficking from the bloodstream, migration within tissues, immune synapse formation, and phagocytosis; and (b) signaling during costimulation and cell polarization. Integrins are so named because they integrate the extracellular and intracellular environments by binding to ligands outside the cell and cytoskeletal components and signaling molecules inside the cell. Integrins are noncovalently associated heterodimeric cell surface adhesion molecules. In vertebrates, 18 α subunits and 8 β subunits form 24 known αβ pairs (Figure 1). This diversity in subunit composition contributes to diversity in ligand recognition, binding to cytoskeletal components and coupling to downstream signaling pathways. Immune cells express at least 10 members of the integrin family belonging to the β2, β7, and β1 subfamilies (Table 1). The β2 and β7 integrins are exclusively expressed on leukocytes, whereas the β1 integrins are expressed on a wide variety of cells throughout the body. Distribution and ligand-binding properties of the integrins on leukocytes are summarized in Table 1. For reviews, see References 1 and 2. Mutations that block expression of the β2 integrin subfamily lead to leukocyte adhesion deficiency, a disease associated with severe immunodeficiency (3).
As adhesion molecules, integrins are unique in that their adhesiveness can be dynamically regulated through a process termed inside-out signaling or priming. Thus, stimuli received by cell surface receptors for chemokines, cytokines, and foreign antigens initiate intracellular signals that impinge on integrin cytoplasmic domains and alter adhesiveness for extracellular ligands. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction (outside-in signaling). These dynamic properties of integrins are central to their proper function in the immune system. Indeed, mutations or small molecules that stabilize either the inactive state or the active adhesive state—and thereby block the adhesive dynamics of leukocyte integrins—inhibit leukocyte migration and normal immune responses.

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Novel Discoveries in Molecular Biology and Biomedical Science

Curator: Larry H. Bernstein, MD, FCAP


Updated June 1, 2016

The following is a collection of current articles on noncoding DNA, synthetic genome engineering, protein regulation of apoptosis, drug design, and geometrics.


No longer ‘junk DNA’ — shedding light on the ‘dark matter’ of the genome

A new tool called “LIGR-Seq” enables scientists to explore in depth what non-coding RNAs actually do in human cells   May 23, 2016

he LIGR-seq method for global-scale mapping of RNA-RNA interactions in vivo to reveal unexpected functions for uncharacterized RNAs that act via base-pairing interactions (credit: University of Toronto)

What used to be dismissed by many as “junk DNA” has now become vitally important, as accelerating genomic data points to the importance of non-coding RNAs (ncRNAs) — a genome’s messages that do not specifically code for proteins — in development and disease.

But our progress in understanding these molecules has been slow because of the lack of technologies that allow for systematic mapping of their functions.

Now, professor Benjamin Blencowe’s team at the University of Toronto’s Donnelly Centre has developed a method called “LIGR-seq” that enables scientists to explore in depth what ncRNAs do in human cells.

The study, described in Molecular Cell, was published on May 19, along with two other papers, in Molecular Cell and Cell, respectively, from Yue Wan’s group at the Genome Institute of Singapore and Howard Chang’s group at Stanford University in California, who developed similar methods to study RNAs in different organisms.

So what exactly do ncRNAs do?

mRNAs vs. ncRNAs (credit: Thomas Shafee/CC)

Of the 3 billion letters in the human genome, only two per cent make up the protein-coding genes. The genes are copied, or transcribed, into messenger RNA (mRNA) molecules, which provide templates for building proteins that do most of the work in the cell. Much of the remaining 98 per cent of the genome was initially considered by some as lacking in functional importance. However, large swaths of the non-coding genome — between half and three quarters of it — are also copied into RNA.

So then what might the resulting ncRNAs do? That depends on whom you ask. Some researchers believe that most ncRNAs have no function, that they are just a by-product of the genome’s powerful transcription machinery that makes mRNA. However, it is emerging that many ncRNAs do have important roles in gene regulation — some ncRNAs act as carriages for shuttling the mRNAs around the cell, or provide a scaffold for other proteins and RNAs to attach to and do their jobs.

But the majority of available data has trickled in piecemeal or through serendipitous discovery. And with emerging evidence that ncRNAs could drive disease progression, such as cancer metastasis, there was a great need for a technology that would allow a systematic functional analysis of ncRNAs.

Up until now, with existing methods, you had to know what you are looking for because they all require you to have some information about the RNA of interest. The power of our method is that you don’t need to preselect your candidates; you can see what’s occurring globally in cells, and use that information to look at interesting things we have not seen before and how they are affecting biology,” says Eesha Sharma, a PhD candidate in Blencowe’s group who, along with postdoctoral fellow Tim Sterne-Weiler, co-developed the method.

A new ncRNA identification tool

The human RNA-RNA interactome, showing interactions detected by LIGR-seq (credit: University of Toronto)

The new ‘‘LIGation of interacting RNA and high-throughput sequencing’’ (LIGR-seq) tool captures interactions between different RNA molecules. When two RNA molecules have matching sequences — strings of letters copied from the DNA blueprint — they will stick together like Velcro. With LIGR-seq, the paired RNA structures are removed from cells and analyzed by state-of-the-art sequencing methods to precisely identify the RNAs that are stuck together.

Most researchers in the life sciences agree that there’s an urgent need to understand what ncRNAs do. This technology will open the door to developing a new understanding of ncRNA function,” says Blencowe, who is also a professor in the Department of Molecular Genetics.

Not having to rely on pre-existing knowledge will boost the discovery of RNA pairs that have never been seen before. Scientists can also, for the first time, look at RNA interactions as they occur in living cells, in all their complexity, unlike in the juices of mashed up cells that they had to rely on before. This is a bit like moving on to explore marine biology from collecting shells on the beach to scuba-diving among the coral reefs, where the scope for discovery is so much bigger.

Actually, ncRNAs come in multiple flavors: there’s rRNA, tRNA, snRNA, snoRNA, piRNA, miRNA, and lncRNA, to name a few, where prefixes reflect the RNA’s place in the cell or some aspect of its function. But the truth is that no one really knows the extent to which these ncRNAs control what goes on in the cell, or how they do this.


Nonetheless, the new technology developed by Blencowe’s group has been able to pick up new interactions involving all classes of RNAs and has already revealed some unexpected findings.

The team discovered new roles for small nucleolar RNAs (snoRNAs), which normally guide chemical modifications of other ncRNAs. It turns out that some snoRNAs can also regulate stability of a set of protein-coding mRNAs. In this way, snoRNAs can also directly influence which proteins are made, as well as their abundance, adding a new level of control in cell biology.

And this is only the tip of the iceberg; the researchers plan to further develop and apply their technology to investigate the ncRNAs in different settings.

“We would like to understand how ncRNAs function during development. We are particularly interested in their role in the formation of neurons. But we will also use our method to discover and map changes in RNA-RNA interactions in the context of human diseases,” says Blencowe.

Abstract of Global Mapping of Human RNA-RNA Interactions

The majority of the human genome is transcribed into non-coding (nc)RNAs that lack known biological functions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked in vivo, “LIGation of interacting RNA followed by high-throughput sequencing” (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.



Venter’s Research Team Creates an Artificial Cell and Reports That 32% of Genes Are Life-Essential but Contain Unknown Functions
May 27, 2016

Understanding the unknown functions of these genes may lead to the creation of new diagnostic tests for clinical laboratories and anatomic pathology groups

Once again, J. Craig Venter, PhD, is charting new ground in gene sequencing andgenomic science. This time his research team has built upon the first synthetic cell they created in 2010 to build a more sophisticated synthetic cell. Their findings from this work may give pathologists and medical laboratory scientists new tools to diagnose disease.

Recently the research team at the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) published their latest findings. Among the things they learned is that science still does not understand the functions of about a third of the genes required for their synthetic cells to function.

JCVI-syn3.0 Could Radically Alter Understanding of Human Genome

Based in La Jolla, Calif., and Rockville, Md., JCVI is a not-for-profit research institute aiming to advance genomics. Building upon its first synthetic cell—Mycoplasma mycoides (M. mycoides) JCVI-syn1.0, which JCVI constructed in 2010—the same team of scientists created the first minimal synthetic bacterial cell, which they calledJCVI-syn3.0. This new artificial cell contains 531,560 base pairs and just 473 genes, which means it is the smallest genome of any organism that can be grown in laboratory media, according to a JCVI-SGI statement.

For pathologists and medical laboratory leaders, the creation of a synthetic life form is a milestone toward better understanding genome sequencing and how this new knowledge may help advance both diagnostics and therapeutics.

“What we’ve done is important because it is a step toward completely understanding how a living cell works,” Clyde Hutchison III, PhD, told New Scientist. “If we can really understand how the cell works, then we will be able to design cells efficiently for the production of pharmaceutical and other useful products.” Hutchison is Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill, Distinguished Professor at the J. Craig Venter Institute, a member of the National Academy of Sciences, and a fellow of the American Academy of Arts and Sciences.

Click here to see images

Clyde Hutchison, III, PhD (above), Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill and Distinguished Professor at the J. Craig Venter Institute, stated that his team’s “goal is to have a cell for which the precise biological function of every gene is known.” (Photo credit: JCVI.)

Understanding a Gene’s True Purpose

According to the JCVI researchers, 149 genes have no known purpose. They are, however, necessary for life and health.

“We know about two-thirds of the essential biology, and we’re missing a third,” stated J. Craig Venter, PhD, Founder and CEO of JCVI, in a story published by MedPage Today.

This knowledge is based upon decades of research. JCVI seeks to create a minimal cell operating system to understand biology, while also providing what the JCVI statement called a “chassis for use in industrial applications.”

What Do these Genes Do Anyway?

The JCVI team found that among most genes’ biological functions:

“JCVI-syn3.0 is a working approximation of a minimal cellular genome—a compromise between a small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life,” the researchers told the journal Science.

More research is needed, the scientists say, into the 149 genes that appear to lack specific biologic functions.

Unlocking Mystery of the 149 Genes Could Lead to Advances in Genomic Science

“Finding so many genes without a known function is unsettling, but it’s exciting because it’s left us with much still to learn. It’s like the ‘dark matter’ of biology,” said Alistair Elfick, PhD, Chair of Synthetic Biological Engineering, University of Edinburgh, UK, in the New Scientist article.

Studies such as JCVI’s research is key to broadening understanding and framing appropriate questions about scientific, ethical, and economic implications of synthetic biology.

The creation of a synthetic cell will have a profound and positive impact on understanding of biology and how life works, JCVI said.

Such research may inspire new whole genome synthesis tools and semi-automated processes that could dramatically affect clinical laboratory procedures. It also could lead to new techniques and tools for advanced vaccine and pharmaceuticals, JCVI pointed out.

—Donna Marie Pocius

Related Information:

First Minimal Synthetic Bacterial Cell Designed and Constructed by Scientists at Venter Institute and Synthetic Genomics, Inc.


CRISPR Versatility Inspires Molecular Biology Innovation

GEN Tech Focus: CRISPR/Gene Editing
No single technique has set the molecular biology field ablaze with excitement and potential like the CRISPR-Cas9 genome editing system has following its introduction only a few short years ago. The following articles represent the flexibility of this technique to potentially treat a host of genetic disorders and possibly even prevent the onset of disease.


CRISPR Moves from Butchery to Surgery

Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.


New CRISPR System Targets Both DNA and RNA

With a staggering number of papers published in the past several years involving the characterization and use of the CRISPR/Cas9 gene editing system, it is surprising that researchers are still finding new features of the versatile molecular scissor enzyme.


High-Fidelity CRISPR-Cas9 Nucleases Virtually Free of Off-Target Noise

If a Cas9 nuclease variant could be engineered that was less grabby, it might loosen its grip on DNA sequences throughout the genome—except those sequences representing on-target sites. That’s the assumption that guided a new investigation by researchers at Massachusetts General Hospital.


CRISPR Works Well but Needs Upgrades

The gene-editing technology known as CRISPR-Cas9 is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice.


A Guide to CRISPR Gene Activation

Published: Tuesday, May 24, 2016
A comparison of synthetic gene-activating Cas9 proteins can help guide research and development of therapeutic approaches.

The CRISPR-Cas9 system has come to be known as the quintessential tool that allows researchers to edit the DNA sequences of many organisms and cell types. However, scientists are also increasingly recognizing that it can be used to activate the expression of genes. To that end, they have built a number of synthetic gene activating Cas9 proteins to study gene functions or to compensate for insufficient gene expression in potential therapeutic approaches.

“The possibility to selectively activate genes using various engineered variants of the CRISPR-Cas9 system left many researchers questioning which of the available synthetic activating Cas9 proteins to use for their purposes. The main challenge was that all had been uniquely designed and tested in different settings; there was no side-by-side comparison of their relative potentials,” said George Church, Ph.D., who is Core Faculty Member at the Wyss Institute for Biologically Inspired Engineering at Harvard University, leader of its Synthetic Biology Platform, and Professor of Genetics at Harvard Medical School. “We wanted to provide that side-by-side comparison to the biomedical research community.”

In a study published on 23 May in Nature Methods, the Wyss Institute team reports how it rigorously compared and ranked the most commonly used artificial Cas9 activators in different cell types from organisms including humans, mice and flies. The findings provide a valuable guide to researchers, allowing them to streamline their endeavors.

The team also included Wyss Core Faculty Member James Collins, Ph.D., who also is the Termeer Professor of Medical Engineering & Science and Professor of Biological Engineering at the Massachusetts Institute of Technology (MIT)’s Department of Biological Engineering and Norbert Perrimon, Ph.D., a Professor of Genetics at Harvard Medical School.

Gene activating Cas9 proteins are fused to variable domains borrowed from proteins with well-known gene activation potentials and engineered so that the DNA editing ability is destroyed. In some cases, the second component of the CRISPR-Cas9 system, the guide RNA that targets the complex to specific DNA sequences, also has been engineered to bind gene-activating factors.

“We first surveyed seven advanced Cas9 activators, comparing them to each other and the original Cas9 activator that served to provide proof-of-concept for the gene activation potential of CRISPR-Cas9. Three of them, provided much higher gene activation than the other candidates while maintaining high specificities toward their target genes,” said Marcelle Tuttle, Research Fellow at the Wyss and a co-lead author of the study.

The team went on to show that the three top candidates were comparable in driving the highest level of gene expression in cells from humans, mice and fruit flies, irrespective of their tissue and developmental origins. The researchers also pinpointed ways to further maximize gene activation employing the three leading candidates.

“In some cases, maximum possible activation of a target gene is necessary to achieve a cellular or therapeutic effect. We managed to cooperatively enhance expression of specific genes when we targeted them with three copies of a top performing activator using three different guide RNAs,” said Alejandro Chavez, Ph.D., a Postdoctoral Fellow and the study’s co-first author.

“The ease of use of CRISPR-Cas9 offers enormous potential for development of genome therapeutics. This study provides valuable new design criteria that will help enable synthetic biologists and bioengineers to develop more effective targeted genome engineering technologies in the future,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.


Engineering T Cells to Functionally Cure HIV-1 Infection

Rachel S Leibman and James L Riley
Molecular Therapy (21 April 2015) |

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients’ lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1–resistant cells, redirecting HIV-1–specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1–specific lymphocytes within a patient’s own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy–mediated functional cure.


Automated top-down design technique simplifies creation of DNA origami nanostructures

Nanoparticles for drug delivery and cell targeting, nanoscale robots, custom-tailored optical devices, and DNA as a storage medium are among the possible applications

May 27, 2016

The boldfaced line, known as a spanning tree, follows the desired geometric shape of the target DNA origami design method, touching each vertex just once. A spanning tree algorithm is used to map out the proper routing path for the DNA strand. (credit: Public Domain)

MITBaylor College of Medicine, and Arizona State University Biodesign Institute researchers have developed a radical new top-down DNA origami* design method based on a computer algorithm that allows for creating designs for DNA nanostructures by simply inputting a target shape.

DNA origami (using DNA to design and build geometric structures) has already proven wildly successful in creating myriad forms in 2- and 3- dimensions, which conveniently self-assemble when the designed DNA sequences are mixed together. The tricky part is preparing the proper DNA sequence and routing design for scaffolding and staple strands to achieve the desired target structure. Typically, this is painstaking work that must be carried out manually.

The new algorithm, which is reported together with a novel synthesis approach in the journal Science, promises to eliminate all that and expands the range of possible applications of DNA origami in biomolecular science and nanotechnology. Think nanoparticles for drug delivery and cell targeting, nanoscale robots in medicine and industry, custom-tailored optical devices, and most interesting: DNA as a storage medium, offering retention times in the millions of years.**


Shape-shifting, top-down software

Unlike traditional DNA origami, in which the structure is built up manually by hand, the team’s radical top-down autonomous design method begins with an outline of the desired form and works backward in stages to define the required DNA sequence that will properly fold to form the finished product.

“The Science paper turns the problem around from one in which an expert designs the DNA needed to synthesize the object, to one in which the object itself is the starting point, with the DNA sequences that are needed automatically defined by the algorithm,” said Mark Bathe, an associate professor of biological engineering at MIT, who led the research. “Our hope is that this automation significantly broadens participation of others in the use of this powerful molecular design paradigm.”

The algorithm, which is known as DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) after the Greek craftsman and artist who designed labyrinths that resemble origami’s complex scaffold structures, can build any type of 3-D shape, provided it has a closed surface. This can include shapes with one or more holes, such as a torus.

A simplified version of the  top-down procedure used to design scaffolded DNA origami nanostructures. It starts with a polygon corresponding to the target shape. Software translates a wireframe version of this structure into a plan for routing DNA scaffold and staple strands. That enables a 3D DNA-based atomic-level structural model that is then validated using 3D cryo-EM reconstruction. (credit: adapted from Biodesign Institute images)

With the new technique, the target geometric structure is first described in terms of a wire mesh made up of polyhedra, with a network of nodes and edges. A DNA scaffold using strands of custom length and sequence is generated, using a “spanning tree” algorithm — basically a map that will automatically guide the routing of the DNA scaffold strand through the entire origami structure, touching each vertex in the geometric form once. Complementary staple strands are then assigned and the final DNA structural model or nanoparticle self-assembles, and is then validated using 3D cryo-EM reconstruction.

The software allows for fabricating a variety of geometric DNA objects, including 35 polyhedral forms (Platonic, Archimedean, Johnson and Catalan solids), six asymmetric structures, and four polyhedra with nonspherical topology, using inverse design principles — no manual base-pair designs needed.

To test the method, simpler forms known as Platonic solids were first fabricated, followed by increasingly complex structures. These included objects with nonspherical topologies and unusual internal details, which had never been experimentally realized before. Further experiments confirmed that the DNA structures produced were potentially suitable for biological applications since they displayed long-term stability in serum and low-salt conditions.

Biological research uses

The research also paves the way for designing nanoscale systems mimicking the properties of viruses, photosynthetic organisms, and other sophisticated products of natural evolution. One such application is a scaffold for viral peptides and proteins for use as vaccines. The surface of the nanoparticles could be designed with any combination of peptides and proteins, located at any desired location on the structure, in order to mimic the way in which a virus appears to the body’s immune system.

The researchers demonstrated that the DNA nanoparticles are stable for more than six hours in serum, and are now attempting to increase their stability further.

The nanoparticles could also be used to encapsulate the CRISPR-Cas9 gene editing tool. The CRISPR-Cas9 tool has enormous potential in therapeutics, thanks to its ability to edit targeted genes. However, there is a significant need to develop techniques to package the tool and deliver it to specific cells within the body, Bathe says.

This is currently done using viruses, but these are limited in the size of package they can carry, restricting their use. The DNA nanoparticles, in contrast, are capable of carrying much larger gene packages and can easily be equipped with molecules that help target the right cells or tissue.

The most exciting aspect of the work, however, is that it should significantly broaden participation in the application of this technology, Bathe says, much like 3-D printing has done for complex 3-D geometric models at the macroscopic scale.

Hao Yan directs the Biodesign Center for Molecular Design and Biomimetics at Arizona State University and is the Milton D. Glick Distinguished Professor, College of Liberal Arts and Sciences, School of Molecular Sciences at ASU.

DNA origami brings the ancient Japanese method of paper folding down to the molecular scale. The basics are simple: Take a length of single-stranded DNA and guide it into a desired shape, fastening the structure together using shorter “staple strands,” which bind in strategic places along the longer length of DNA. The method relies on the fact that DNA’s four nucleotide letters—A, T, C, & G stick together in a consistent manner — As always pairing with Ts and Cs with Gs.

The DNA molecule in its characteristic double stranded form is fairly stiff, compared with single-stranded DNA, which is flexible. For this reason, single stranded DNA makes for an ideal lace-like scaffold material. Further, its pairing properties are predictable and consistent (unlike RNA).

** A single gram of DNA can store about 700 terabytes of information — an amount equivalent to 14,000 50-gigabyte Blu-ray disks — and could potentially be operated with a fraction of the energy required for other information storage options.


Essential role of miRNAs in orchestrating the biology of the tumor microenvironment

Jamie N. Frediani and Muller Fabbri
Molecular Cancer (2016) 15:42

MicroRNAs (miRNAs) are emerging as central players in shaping the biology of the Tumor Microenvironment (TME). They do so both by modulating their expression levels within the different cells of the TME and by being shuttled among different cell populations within exosomes and other extracellular vesicles. This review focuses on the state-of-the-art knowledge of the role of miRNAs in the complexity of the TME and highlights limitations and challenges in the field. A better understanding of the mechanisms of action of these fascinating micro molecules will lead to the development of new therapeutic weapons and most importantly, to an improvement in the clinical outcome of cancer patients. Keywords: Exosomes, microRNAs, Tumor microenvironment, Cancer

While cancer treatment and survival have improved worldwide, the need for further understanding of the underlying tumor biology remains. In recent years, there has been a significant shift in scientific focus towards the role of the tumor microenvironment (TME) on the development, growth, and metastatic spread of malignancies. The TME is defined as the surrounding cellular environment enmeshed around the tumor cells including endothelial cells, lymphocytes, macrophages, NK cells, other cells of the immune system, fibroblasts, mesenchymal stem cells (MSCs), and the extracellular matrix (ECM). Each of these components interacts with and influences the tumor cells, continually shifting the balance between pro- and anti-tumor phenotype. One of the predominant methods of communication between these cells is through extracellular vesicles and their microRNA (miRNA) cargo. Extracellular vesicles (EVs) are between 30 nm to a few microns in diameter, are surrounded by a phospholipid bilayer membrane, and are released from a variety of cell types into the local environment. There are three well characterized groups of EVs: 1) exosomes, typically 30–100 nm, 2) microvesicles (or ectosomes), typically 100–1000 nm, and 3) large oncosomes, typically 1–10 μm. Each of these categories has a distinctly unique biogenesis and purpose in cellcell communication despite the fact that current laboratory methods do not always allow precise differentiation. EVs are found to be enriched with membrane-bound proteins, lipid raft-associated and cytosolic proteins, lipids, DNA, mRNAs, and miRNAs, all of which can be transferred to the recipient cell upon fusion to allow cell-cell communications [1]. Of these, miRNAs have been of particular interest in cancer research, both as modifiers of transcription and translation as well as direct inhibitors or enhancers of key regulatory proteins. These miRNAs are a large family of small non-coding RNAs (19–24 nucleotides) and are known to be aberrantly expressed, both in terms of content as well as number, in both the tumor cells and the cells of the TME. Synthesis of these mature miRNA is a complex process, starting with the transcription of long, capped, and polyadenylated pri-miRNA by RNA polymerase II. These are cropped into a 60–100 nucleotide hairpinstructure pre-miRNA by the microprocessor, a heterodimer of Drosha (a ribonuclease III enzyme) and DGCR8 (DiGeorge syndrome critical region gene 8). The premiRNA is then exported to the cytoplasm by exportin 5, cleaved by Dicer, and separated into single strands by helicases. The now mature miRNA are incorporated into the RNA-induced silencing complex (RISC), a cytoplasmic effector machine of the miRNA pathway. The primary mechanism of action of the mature miRNA-RISC complex is through their binding to the 3’ untranslated region, or less commonly the 5’ untranslated region, of target mRNA, leading to protein downregulation either via translational repression or mRNA degradation. More recently, it has been shown that miRNAs can also upregulate the expression of target genes [2]. MiRNA genes are mostly intergenic and are transcribed by independent promoters [3] but can also be encoded by introns, sharing the same promoter of their host gene [4]. MiRNAs undergo the same regulatory mechanisms of any other protein coding gene (promoter methylation, histone modifications, etc.…) [5, 6]. Interestingly, each miRNA may have contradictory effects both within varying tumor cell lines and within different cells of the TME. In this review, we provide a state-of-the-art description of the key role that miRNAs have in the communication between tumor cells and the TME and their subsequent effects on the malignant phenotype. Finally, this review has made every effort to clarify, whenever possible, whether the reference is to the −3p or the -5p miRNA. Whenever such clarification has not been provided, this indicates that it was not possible to infer such information from the cited bibliography.

Angiogenesis and miRNAs Cellular plasticity, critical in the development of malignancy, includes the many diverse mechanisms elicited by cancer cells to increase their malignant potential and develop increasing treatment resistance. One such mechanism, angiogenesis, is critical to the development of metastatic disease, affecting both the growth of malignant cells locally and their survival at distant sites. In the last ten years, miRNAs, often packaged in tumor cell-derived exosomes, have emerged as important contributors to the complicated regulation and balance of pro- and anti-angiogenic factors.

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7].

Fig. 1

Central role of the miR-17-92 cluster in the biology of the TME. The miR-17-92 cluster encoding miR-17, −18a, −19b, −20a, and -92a is upregulated in multiple tumor types and interacts with various components of the TME to finely “tune” the TME through a complex combination of pro- and anti-tumoral effects

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7]. Up-regulation of this particular locus has further been confirmed in miRnome analysis across multiple different tumor types, including lung, breast, stomach, prostate, colon, and pancreatic cancer [8]. The miR-17-92 cluster is directly activated by Myc and modulates a variety of downstream transcription factors important in cell cycle regulation and apoptosis including activation of E2F family and Cyclin-dependent kinase inhibitor (CDKN1A) and downregulation of BCL2L11/BIM and p21 [7]. In addition to promoting cell cycle progression and inhibiting apoptosis, the miR-17-92 cluster also downregulates thrombospondin-1 (Tsp1) and connective tissue growth factor (CTGF), important antiangiogenic proteins [7]. Similarly, microvesicles from colorectal cancer cells contain miR-1246 and TGF-β which are transferred to endothelial cells to silence promyelocytic leukemia protein (PML) and activate Smad 1/5/8 signaling promoting proliferation and migration [9]. Likewise, lung cancer cell line derived microvesicles contain miR-494, in response to hypoxia, which targets PTEN in the endothelial cells promoting angiogenesis through the Akt/eNOS pathway [10]. Lastly, exosomal miR-135b from multiple myeloma cells suppresses the HIF-1/FIH-1 pathway in endothelial cells, increasing angiogenesis [11]. A summary of the studies showing the functions of exosomal miRNAs in shaping the biology of the TME is provided in Table 1.


Table 1

Actions of exosomal miRNAs exchanged between cells of the TME




Cell of origin

Accepting cell


Effect on TME



Multiple myeloma

Endothelial cells





Lung cancer

Endothelial cells





Endothelial cells

Breast cancer

Cyclin D2 and D3

↓Tumor growth and invasion



Colorectal cancer

Endothelial Cells

PML/Smad 1/5/8

↑ Growth & migration


Stromal compartment:


Breast cancer

Endothelial cells


↓Tight junctions

↑Metastatic progression




Stromal cells


↑Tumor growth


Immune system:





↑Growth & metastasis






↑Growth & metastasis











↑ Drug resistance



Hypoxic tumor derived

NK cells


↓ NK cell response




Tumor cells (various)

Regulatory T cells






Breast cancer


↑ Invasion


Abbreviations: TAMs Tumor Associated Macrophages, CLL chronic lymphocytic leukemia, NSCLCnon-small cell lung cancer, NBL Neuroblastoma

The most common target of anti-angiogenic therapy is VEGF, and not unsurprisingly, multiple miRNAs (including miR-9, miR-20b, miR-130, miR-150, and miR-497) promote angiogenesis through the induction of the VEGF pathway. The most studied of these is the up-regulation of miR-9 which has been linked to a poor prognosis in multiple tumor types, including breast cancer, non-small cell lung cancer, and melanoma [12]. The two oncogenes MYC and MYCN activate miR-9 and cause E-cadherin downregulation resulting in the upregulated transcription of VEGF [13]. In addition, miR-9 has been shown to upregulate the JAK-STAT pathway, supporting endothelial cell migration and tumor angiogenesis [13]. Both amplification of miR-20b and miR-130 as well as miR-497 suppression regulate VEGF through hypoxia inducible factor 1α (HIF-1α) supporting increased angiogenesis [14, 15, 16, 17]. …..

The pivotal discovery in 2012 by Mitra et al. laid the ground-work for our current knowledge on the interactions between tumor-derived miRNAs and fibroblasts. In combination, the down-regulation of miR-214 and miR-31 and the up-regulation of miR-155 trigger the reprogramming of quiescent fibroblasts to CAFs [32]. As expected, the reverse regulation of these miRNAs reduced the migration and invasion of co-cultured ovarian cancer cells [32]. While the pathway of miR-155’s involvement in CAF biology is still being elucidated, the pathways of miR-214 and miR-31 have been established. In endometrial cancer, miR-31 was found to target the homeobox gene SATB2, leading to enhanced tumor cell migration and invasion [33]. MiR-214 similarly has an inverse correlation with its chemokine target, C-C motif Ligand 5 (CCL5) [32]. CCL5 secretion has been associated with enhanced motility, invasion, and metastatic potential through NF-κB-mediated MMP9 activation and through generation and differentiation of myeloid-derived suppressor cells (MDSCs) [34, 35, 36]. Furthermore, miR-210 and miR-133b overexpression and miR-149 suppression have been subsequently found to independently trigger the conversion to CAFs, possibly through paracrine stimulation, and to additionally promote EMT in prostate and gastric cancer, respectively [37, 38,39]. MiR-210 additionally enlists monocytes and encourages angiogenesis [37].   …

Another function of CAFs is the destruction of the ECM and its remodeling with a tumor-supportive composition and structure which includes modulation of specific integrins and metalloproteinases as some of the most studied miRNA targets. The 23 matrix metalloproteinases (MMPs) are critical in the ECM degradation, disruption of the growth signal balance, resistance to apoptosis, establishment of a favorable metastatic niche, and promotion of angiogenesis [54]. As expected, miRNAs have been found to regulate the actions of MMPs, together working to promote cancer cell growth, invasiveness, and metastasis. In HCC, MMP2 and 9 expression is up-regulated by miR-21 via PTEN pathway downregulation. Similarly, in cholangiocarcinoma it was observed that reduced levels of miR-138 induced up-regulation of RhoC, leading to increased levels of the same two MMPs [55, 56]. ….

As has been shown throughout this review, miRNAs have an important and varied effect on human carcinogenesis by shaping the biology of the TME towards a more permissive pro-tumoral phenotype. The complex events leading to such an outcome are currently quite universally defined as the “educational” process of cancer cells on the surrounding TME. While the initial focus was on the direction from the cancer cell to the surrounding TME, increasingly interest is centered on the implications of a more dynamic bidirectional exchange of genetic information. MiRNAs represent only part of the cargo of the extracellular vesicles, but an increasing scientific literature points towards their pivotal role in creating the micro-environmental conditions for cancer cell growth and dissemination. The nearby future will have to address several questions still unanswered. First, it is absolutely necessary to clarify which miRNAs and to what extent they are involved in this process. The contradictory results of some studies can be explained by the differences in tumor-types and by different concentrations of miRNAs used for functional studies. Understanding whether different concentrations of the same miRNA elicit different target effects and therefore changes the biology of the TME, will represent a significant consideration in the development of this field. It is certainly very attractive (especially in an attempt to develop new and desperately needed better cancer biomarkers) to think that concentrations of miRNAs within the TME are reflected systemically in the circulating levels of that same miRNA, however this has not yet been irrefutably demonstrated. Moreover, the study of the paracrine interactions among different cell populations of the TME and their reciprocal effects has been limited to two, maximum three cell populations. This is still way too far from describing the complexity of the TME and only the development of new tridimensional models of the TME will be able to cast a more conclusive light on such complexity. Finally, the pharmacokinetics of miRNA-containing vesicles is in its infancy at best, and needs to be further developed if the goal is development of new therapies based on the use of exosomic miRNAs. Therefore, the future of miRNA research, particularly in its role in the TME, holds still a lot of questions that need answering. However, for these exact same reasons, this is an incredibly exciting time for research in this field. We can envision a not too far future in which these concerns will be satisfactorily addressed and our understanding of the role of miRNAs within the TME will allow us to use them as new therapeutic weapons to successfully improve the clinical outcome of cancer patients.




Triggering the protein that programs cancer cells to kill themselves

May 24, 2016
WEHI | Apoptosis

Researchers at the Walter and Eliza Hall Institute in Australia have discovered a new way to trigger cell death that could lead to drugs to treat cancer and autoimmune disease.

Programmed cell death (a.k.a. apoptosis) is a natural process that removes unwanted cells from the body. Failure of apoptosis can allow cancer cells to grow unchecked or immune cells to inappropriately attack the body.

The protein known as Bak is central to apoptosis. In healthy cells, Bak sits in an inert state but when a cell receives a signal to die, Bak transforms into a killer protein that destroys the cell.

Triggering the cancer-apoptosis trigger

Institute researchers Sweta Iyer, PhD, Ruth Kluck, PhD, and colleagues unexpectedly discovered that an antibody they had produced to study Bak actually bound to the Bak protein and triggered its activation. They hope to use this discovery to develop drugs that promote cell death.

The researchers used information about Bak’s three-dimensional structure to find out precisely how the antibody activated Bak. “It is well known that Bak can be activated by a class of proteins called ‘BH3-only proteins’ that bind to a groove on Bak. We were surprised to find that despite our antibody binding to a completely different site on Bak, it could still trigger activation,” Kluck said.  “The advantage of our antibody is that it can’t be ‘mopped up’ and neutralized by pro-survival proteins in the cell, potentially reducing the chance of drug resistance occurring.”

Drugs that target this new activation site could be useful in combination with other therapies that promote cell death by mimicking the BH3-only proteins. The researchers are now working with collaborators to develop their antibody into a drug that can access Bak inside cells.

Their findings have just been published in the open-access journal Nature Communications. The research was supported by the National Health and Medical Research Council, the Australian Research Council, the Victorian State Government Operational Infrastructure Support Scheme, and the Victorian Life Science Computation Initiative.

Abstract of Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.



Catching metastatic cancer cells before they grow into tumors: a new implant shows promise

Cure” is a word that’s dominated the rhetoric in the war on cancer for decades. But it’s a word that medical professionals tend to avoid. While the American Cancer Society reports that cancer treatment has improved markedly over the decades and the five-year survival rate is impressively high for many cancers, oncologists still refrain from declaring their cancer-free patients cured. Why?

Patients are declared cancer-free (also called complete remission) when there are no more signs of detectable disease.

However, minuscule clusters of cancer cells below the detection level can remain in a patient’s body after treatment. Moreover, such small clusters of straggler cells may undergo metastasis, where they escape from the initial tumor into the bloodstream and ultimately settle in a distant site, often a vital organ such as the lungs, liver or brain.

Cancer cells can move throughout the body, like these metastatic melanoma cells. NIH Image Gallery/FlickrCC BY

When a colony of these metastatic cells reaches a detectable size, the patient is diagnosed with recurrent metastatic cancer. About one in three breast cancer patients diagnosed with early-stage cancer later develop metastatic disease, usually within five years of initial remission.

By the time metastatic cancer becomes evident, it is much more difficult to treat than when it was originally diagnosed.

What if these metastatic cells could be detected earlier, before they established a “foothold” in a vital organ? Better yet, could these metastatic cancer cells be intercepted, preventing them them from lodging in a vital organ in the first place?

To catch a cancer cell

With these goals in mind, our biomaterials lab joined forces with surgical oncologist Jacqueline Jeruss to create an implantable medical device that acts as a metastatic cancer cell trap.

The implant is a tiny porous polymer disc (basically a miniature sponge, no larger than a pencil eraser) that can be inserted just under a patient’s skin. Implantation triggers the immune system’s “foreign body response,” and the implant starts to soak up immune cells that travel to it. If the implant can catch mobile immune cells, then why not mobile metastatic cancer cells?

The disc can detect cancer cells in mice. Lab mouse via

We gave implants to mice specially bred to model metastatic breast cancer. When the mice had palpable tumors but no evidence of metastatic disease, the implant was removed and analyzed.

Cancer cells were indeed present in the implant, while the other organs (potential destinations for metastatic cells) still appeared clean. This means that the implant can be used to spot previously undetectable metastatic cancer before it takes hold in an organ.

For patients with cancer in remission, an implant that can detect tumor cells as they move through the body would be a diagnostic breakthrough. But having to remove it to see if it has captured any cancer cells is not the most convenient or pleasant detection method for human patients.

Detecting cancer cells with noninvasive imaging

There could be a way around this, though: a special imaging method under development at Northwestern University called Inverse Spectroscopic Optical Coherence Tomography (ISOCT). ISOCT detects molecular-level differences in the way cells in the body scatter light. And when we scan our implant with ISOCT, the light scatter pattern looks different when it’s full of normal cells than when cancer cells are present. In fact, the difference is apparent when even as few as 15 out of the hundreds of thousands of cells in the implant are cancer cells.

There’s a catch – ISOCT cannot penetrate deep into tissue. That means it is not a suitable imaging technology for finding metastatic cells buried deep in internal organs. However, when the cancer cell detection implant is located just under the skin, it may be possible to detect cancer cells trapped in it using ISOCT. This could offer an early warning sign that metastatic cells are on the move.

This early warning could prompt doctors to monitor their patients more closely or perform additional tests. Conversely, if no cells are detected in the implant, a patient still in remission could be spared from unneeded tests.

The ISOCT results show that noninvasive imaging of the implant is feasible. But it’s a method still under development, and thus it’s not widely available. To make scanning easier and more accessible, we’re working to adapt more ubiquitous imaging technologies like ultrasound to detect tiny quantities of tumor cells in the implant.

Detect and capture. Joseph Xu, Michigan EngineeringCC BY-NC-ND

Not just detecting, but quarantining cancer

Besides providing a way to detect tiny numbers of cancer cells before they can form new tumors in other parts of the body, our implant offers an even more intriguing possibility: diverting metastatic cells away from vital organs, and sequestering them where they cannot cause any damage.

In our mouse studies, we found that metastatic cells got caught in the implant before they were apparent in vital organs. When metastatic cells eventually made their way into the organs, the mice with implants still had significantly fewer tumor cells in their organs than implant-free controls. Thus, the implant appears to provide a therapeutic benefit, most likely by taking the metastatic cells it catches out of the circulation, preventing them from lodging anywhere vital.

Interestingly, we have not seen cancer cells leave the implant once trapped, or form a secondary tumor in the implant. Ongoing work aims to learn why this is. Whether the cells can stay safely immobilized in the implant or if it would need to be removed periodically will be important questions to answer before the implant could be used in human patients.

What the future may hold

For now, our work aims to make the implant more effective at drawing and detecting cancer cells. Since we tested the implant with metastatic breast cancer cells, we also want to see if it will work on other types of cancer. Additionally, we’re studying the cells the implant traps, and learning how the implant interacts with the body as a whole. This basic research should give us insight into the process of metastasis and how to treat it.

In the future (and it might still be far off), we envision a world where recovering cancer patients can receive a detector implant to stand guard for disease recurrence and prevent it from happening. Perhaps the patient could even scan their implant at home with a smartphone and get treatment early, when the disease burden is low and the available therapies may be more effective. Better yet, perhaps the implant could continually divert all the cancer cells away from vital organs on its own, like Iron Man’s electromagnet that deflects shrapnel from his heart.

This solution is still not a “cure.” But it would transform a formidable disease that one out of three cancer survivors would otherwise ultimately die from into a condition with which they could easily live.


New PSA Test Examines Protein Structures to Detect Prostate Cancers

5/16/2016  by Cleveland Clinic

A promising new test is detecting prostate cancer more precisely than current tests, by identifying molecular changes in the prostate specific antigen (PSA) protein, according to Cleveland Clinic research presented today at the American Urological Association annual meeting.

The study – part of an ongoing multicenter prospective clinical trial – found that the IsoPSATM test can also differentiate between high-risk and low-risk disease, as well as benign conditions.

Although widely used, the current PSA test relies on detection strategies that have poor specificity for cancer – just 25 percent of men who have a prostate biopsy due to an elevated PSA level actually have prostate cancer, according to the National Cancer Institute – and an inability to determine the aggressiveness of the disease.

The IsoPSA test, however, identifies prostate cancer in a new way. Developed by Cleveland Clinic, in collaboration with Cleveland Diagnostics, Inc., IsoPSA identifies the molecular structural changes in protein biomarkers. It is able to detect cancer by identifying these structural changes, as opposed to current tests that simply measure the protein’s concentration in a patient’s blood.

“While the PSA test has undoubtedly been one of the most successful biomarkers in history, its limitations are well known. Even currently available prostate cancer diagnostic tests rely on biomarkers that can be affected by physiological factors unrelated to cancer,” said Eric Klein, M.D., chair of Cleveland Clinic’s Glickman Urological & Kidney Institute. “These study results show that using structural changes in PSA protein to detect cancer is more effective and can help prevent unneeded biopsies in low-risk patients.”

The clinical trial involves six healthcare institutions and 132 patients, to date. It examined the ability of IsoPSA to distinguish patients with and without biopsy-confirmed evidence of cancer. It also evaluated the test’s precision in differentiating patients with high-grade (Gleason = 7) cancer from those with low-grade (Gleason = 6) disease and benign findings after standard ultrasound-guided biopsy of the prostate.

Substituting the IsoPSA structure-based composite index for the standard PSA resulted in improvement in diagnostic accuracy. Compared with serum PSA testing, IsoPSA performed better in both sensitivity and specificity.

“We took an ‘out of the box’ approach that has shown success in detecting prostate cancer but also has the potential to address other clinically important questions such as clinical surveillance of patients after treatment,” said Mark Stovsky, M.D., staff member, Cleveland Clinic Glickman Urological & Kidney Institute’s Department of Urology. Stovsky has a leadership position (Chief Medical Officer) and investment interest in Cleveland Diagnostics, Inc. “In general, the clinical utility of prostate cancer early detection and screening tests is often limited by the fact that biomarker concentrations may be affected by physiological processes unrelated to cancer, such as inflammation, as well as the relative lack of specificity of these biomarkers to the cancer phenotype. In contrast, clinical research data suggests that the IsoPSA assay can interrogate the entire PSA isoform distribution as a single stand-alone diagnostic tool which can reliably identify structural changes in the PSA protein that correlate with the presence or absence and aggressiveness of prostate cancer.”


Point of Care, Highly Accurate Cervical Cancer Screening

5/20/2016 by Avi Rosenzweig, VP of Business Development, Biop Medical

Fifty-five million times a year, American women go to their gynecologist for a Pap Smear. After waiting a few weeks for the results, more than 3.5 million of them are called back to the physician for a follow up visualization of the cervix. Beyond the stress related to possibly having cancer, the women are then subjected to a colposcopic exam, and all too often, a painful biopsy. Then more stressful waiting for a final diagnosis from the pathologist.

Cervical cancer develops slowly, allowing for successful treatment, when identified on time. Regions with high screening compliancy have low mortality rates from this cancer. In the US, for instance, where screening rates are close to 90%, only 4,200 women die from cervical cancer, annually, or 2.6 women per 100,000. However, the screening process in the developed world is long, complicated and not optimized.

In developing regions however, cervical cancer is a leading cause of women death. Over 85% of the total deaths from this cancer are in developing countries. Regions suffering from low screening rates include not only Africa, India and China, but many Eastern European countries as well. According to an OECD report from 2014, the cervical cancer screening rates in Romania and Hungary are as low as 14.6% and 36.7% respectively. The mortality rates in these countries are high, 16 in 100,000 women in Romania and 7.7 in 100,000 in Hungary.

The current screening process for cervical cancer detection is long, beginning with a Pap or HPV test. Cytology results take weeks to receive. A positive result requires follow-up testing by colposcopy and often biopsy. In countries where there is little access to medical care, or where screening compliancy is low, the chances of successful detection via this multi-step process are small. Developing regions and non-compliant countries require a point of care diagnostic method, which eliminates the need for return visits.

Additional limitations to cervical cancer screening are the low sensitivity and specificity rates of Pap tests and the high false positive rates of HPV test, leading to unnecessary colposcopies. Both cytology and colposcopy testing are highly dependent on operator proficiency for accurate diagnosis.

Biop has developed a new technology for the optimization of this process, into one, three minute, painless optical scan. The vaginal probe uses advanced optical, imaging and non-imaging technologies to identify and classify epithelium based cancers and pre-cancerous lesions. The probe is inserted into the vaginal canal, and scans the entire cervix. The resulting images and optical signatures created from the light, and captured by the sensors, are analyzed by the proprietary algorithm. The result is two pictures, on the physician’s screen; a high resolution photograph of the patient’s cervix, immediately next to a hot/cold map indicating a precise classification and location of any diseased lesions.


Deep learning applied to drug discovery and repurposing

May 27, 2016

Deep neural networks for drug discovery (credit: Insilico Medicine, Inc.)

Scientists from Insilico Medicine, Inc. have trained deep neural networks (DNNs) to predict the potential therapeutic uses of 678 drugs, using gene-expression data obtained from high-throughput experiments on human cell lines from Broad Institute’s LINCS databases and NIH MeSH databases.

The supervised deep-learning drug-discovery engine used the properties of small molecules, transcriptional data, and literature to predict efficacy, toxicity, tissue-specificity, and heterogeneity of response.

“We used LINCS data from Broad Institute to determine the effects on cell lines before and after incubation with compounds, co-author and research scientist Polina Mamoshina explained to KurzweilIAI.

“We used gene expression data of total mRNA from cell lines extracted and measured before incubation with compound X and after incubation with compound X to identify the response on a molecular level. The goal is to understand how gene expression (the transcriptome) will change after drug uptake. It is a differential value, so we need a reference (molecular state before incubation) to compare.”

The research is described in a paper in the upcoming issue of the journal Molecular Pharmaceutics.

Helping pharmas accelerate R&D

Alex Zhavoronkov, PhD, Insilico Medicine CEO, who coordinated the study, said the initial goal of their research was to help pharmaceutical companies significantly accelerate their R&D and increase the number of approved drugs. “In the process we came up with more than 800 strong hypotheses in oncology, cardiovascular, metabolic, and CNS spaces and started basic validation,” he said.

The team measured the “differential signaling pathway activation score for a large number of pathways to reduce the dimensionality of the data while retaining biological relevance.” They then used those scores to train the deep neural networks.*

“This study is a proof of concept that DNNs can be used to annotate drugs using transcriptional response signatures, but we took this concept to the next level,” said Alex Aliper, president of research, Insilico Medicine, Inc., lead author of the study.

Via Pharma.AI, a newly formed subsidiary of Insilico Medicine, “we developed a pipeline for in silico drug discovery — which has the potential to substantially accelerate the preclinical stage for almost any therapeutic — and came up with a broad list of predictions, with multiple in silico validation steps that, if validated in vitro and in vivo, can almost double the number of drugs in clinical practice.”

Despite the commercial orientation of the companies, the authors agreed not to file for intellectual property on these methods and to publish the proof of concept.

Deep-learning age biomarkers

According to Mamoshina, earlier this month, Insilico Medicine scientists published the first deep-learned biomarker of human age — aiming to predict the health status of the patient — in a paper titled “Deep biomarkers of human aging: Application of deep neural networks to biomarker development” by Putin et al, in Aging; and an overview of recent advances in deep learning in a paper titled “Applications of Deep Learning in Biomedicine” by Mamoshina et al., also in Molecular Pharmaceutics.

Insilico Medicine is located in the Emerging Technology Centers at Johns Hopkins University in Baltimore, Maryland, in collaboration with Datalytic Solutions and Mind Research Network.

* In this study, scientists used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the Library of Integrated Network-Based Cellular Signatures (LINCS) project developed by the National Institutes of Health (NIH) and linked those to 12 therapeutic use categories derived from MeSH (Medical Subject Headings) developed and maintained by the National Library of Medicine (NLM) of the NIH.

To train the DNN, scientists utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. Cross-validation experiments showed that DNNs achieve 54.6% accuracy in correctly predicting one out of 12 therapeutic classes for each drug.

One peculiar finding of this experiment was that a large number of drugs misclassified by the DNNs had dual use, suggesting possible application of DNN confusion matrices in drug repurposing.
FutureTechnologies Media Group | Video presentation Insilico medicine

Abstract of Deep learning applications for predicting pharmacological properties of drugs and drug repurposing using transcriptomic data

Deep learning is rapidly advancing many areas of science and technology with multiple success stories in image, text, voice and video recognition, robotics and autonomous driving. In this paper we demonstrate how deep neural networks (DNN) trained on large transcriptional response data sets can classify various drugs to therapeutic categories solely based on their transcriptional profiles. We used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the LINCS project and linked those to 12 therapeutic use categories derived from MeSH. To train the DNN, we utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. When applied to normalized gene expression data for “landmark genes,” DNN showed cross-validation mean F1 scores of 0.397, 0.285 and 0.234 on 3-, 5- and 12-category classification problems, respectively. At the pathway level DNN performed best with cross-validation mean F1 scores of 0.701, 0.596 and 0.546 on the same tasks. In both gene and pathway level classification, DNN convincingly outperformed support vector machine (SVM) model on every multiclass classification problem. For the first time we demonstrate a deep learning neural net trained on transcriptomic data to recognize pharmacological properties of multiple drugs across different biological systems and conditions. We also propose using deep neural net confusion matrices for drug repositioning. This work is a proof of principle for applying deep learning to drug discovery and development.



Transistor-based biosensor detects molecules linked to cancer, Alzheimer’s, and Parkinson’s

May 23, 2016

An inexpensive portable biosensor developed by researchers at Brazil’s National Nanotechnology Laboratory (credit: LNNano)

A novel nanoscale organic transistor-based biosensor that can detect molecules associated with neurodegenerative diseases and some types of cancer has been developed by researchers at the National Nanotechnology Laboratory (LNNano) in Brazil.

The transistor, mounted on a glass slide, contains the reduced form of the peptide glutathione (GSH), which reacts in a specific way when it comes into contact with the enzyme glutathione S-transferase (GST), linked to Parkinson’s, Alzheimer’s and breast cancer, among other diseases.

Sensitive water-gated copper phthalocyanine (CuPc) thin-film transistor (credit: Rafael Furlan de Oliveira et al./Organic Electronics)

“The device can detect such molecules even when they’re present at very low levels in the examined material, thanks to its nanometric sensitivity,” explained Carlos Cesar Bof Bufon, Head of LNNano’s Functional Devices & Systems Lab (DSF).

Bufon said the system can be adapted to detect other substances by replacing the analytes (detection compounds). The team is working on paper-based biosensors to further lower the cost, improve portability, and facilitate fabrication and disposal.

The research is published in the journal Organic Electronics.

Abstract of Water-gated phthalocyanine transistors: Operation and transduction of the peptide–enzyme interaction

The use of aqueous solutions as the gate medium is an attractive strategy to obtain high charge carrier density (1012 cm−2) and low operational voltages (<1 V) in organic transistors. Additionally, it provides a simple and favorable architecture to couple both ionic and electronic domains in a single device, which is crucial for the development of novel technologies in bioelectronics. Here, we demonstrate the operation of transistors containing copper phthalocyanine (CuPc) thin-films gated with water and discuss the charge dynamics at the CuPc/water interface. Without the need for complex multilayer patterning, or the use of surface treatments, water-gated CuPc transistors exhibited low threshold (100 ± 20 mV) and working voltages (<1 V) compared to conventional CuPc transistors, along with similar charge carrier mobilities (1.2 ± 0.2) x 10−3 cm2 V−1 s−1. Several device characteristics such as moderate switching speeds and hysteresis, associated with high capacitances at low frequencies upon bias application (3.4–12 μF cm−2), indicate the occurrence of interfacial ion doping. Finally, water-gated CuPc OTFTs were employed in the transduction of the biospecific interaction between tripeptide reduced glutathione (GSH) and glutathione S-transferase (GST) enzyme, taking advantage of the device sensitivity and multiparametricity.



First Large-Scale Proteogenomic Study of Breast Cancer    

Tues, May 31, 2016

The study offers understanding of potential therapeutic targets.

Building on data from The Cancer Genome Atlas (TCGA) project, a multi-institutional team of scientists have completed the first large-scale “proteogenomic” study of breast cancer, linking DNA mutations to protein signaling and helping pinpoint the genes that drive cancer. Conducted by members of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), including Baylor College of Medicine, Broad Institute of MIT and Harvard, Fred Hutchinson Cancer Research Center, New York University Langone Medical Center, and Washington University School of Medicine, the study takes aim at proteins, the workhorses of the cell, and their modifications to better understand cancer.

Appearing in the Advance Online Publication of Nature, the study illustrates the power of integrating genomic and proteomic data to yield a more complete picture of cancer biology than either analysis could do alone. The effort produced a broad overview of the landscape of the proteome (all the proteins found in a cell) and the phosphoproteome (the sites at which proteins are tagged by phosphorylation, a chemical modification that drives communication in the cell) across a set of 77 breast cancer tumors that had been genomically characterized in the TCGA project. Although the TCGA produced an extensive catalog of somatic mutations found in cancer, the effects of many of those mutations on cellular functions or patients’ outcomes are unknown.

In addition, not all mutated genes are true “drivers” of cancer — some are merely “passenger” mutations that have little functional consequence. And some mutations are found within very large DNA regions that are deleted or present in extra copies, so winnowing the list of candidate genes by studying the activity of their protein products can help identify therapeutic targets. “We don’t fully understand how complex cancer genomes translate into the driving biology that causes relapse and mortality,” said Matthew Ellis, director of the Lester and Sue Smith Breast Center at Baylor College of Medicine and a senior author of the paper.

“These findings show that proteogenomic integration could one day prove to be a powerful clinical tool, allowing us to traverse the large knowledge gap between cancer genomics and clinical action.” In this study, the researchers at the Broad Institute analyzed breast tumors using accurate mass, high-resolution mass spectrometry, a technology that extends the coverage of the proteome far beyond the coverage that can be achieved by traditional antibody-based methods. This allowed them to scale their efforts and quantify more than 12,000 proteins and 33,000 phosphosites, an extremely deep level of coverage.


Breakthrough Approach to Breast Cancer Treatment

May 24, 2016

Scripps scientists have designed a drug candidate that decreases growth of breast cancer cells.

In a development that could lead to a new generation of drugs to precisely treat a range of diseases, scientists from the Florida campus of The Scripps Research Institute (TSRI) have for the first time designed a drug candidate that decreases the growth of tumor cells in animal models in one of the hardest to treat cancers—triple negative breast cancer.

“This is the first example of taking a genetic sequence and designing a drug candidate that works effectively in an animal model against triple negative breast cancer,” said TSRI Professor Matthew Disney. “The study represents a clear breakthrough in precision medicine, as this molecule only kills the cancer cells that express the cancer-causing gene—not healthy cells. These studies may transform the way the lead drugs are identified—by using the genetic makeup of a disease.”

The study, published by the journal Proceedings of the National Academy of Sciences, demonstrates that the Disney lab’s compound, known as Targaprimir-96, triggers breast cancer cells to kill themselves via programmed cell death by precisely targeting a specific RNA that ignites the cancer.

Short-Cut to Drug Candidates

While the goal of precision medicine is to identify drugs that selectively affect disease-causing biomolecules, the process has typically involved time-consuming and expensive high-throughput screens to test millions of potential drug candidates to identify those few that affect the target of interest. Disney’s approach eliminates these screens.

The new study uses the lab’s computational approach called Inforna, which focuses on developing designer compounds that bind to RNA folds, particularly microRNAs.

MicroRNAs are short molecules that work within all animal and plant cells, typically functioning as a “dimmer switch” for one or more genes, binding to the transcripts of those genes and preventing protein production. Some microRNAs have been associated with diseases. For example, microRNA-96, which was the target of the new study, promotes cancer by discouraging programmed cell death, which can rid the body of cells that grow out of control.

In the new study, the drug candidate was tested in animal models over a 21-day course of treatment. Results showed decreased production of microRNA-96 and increased programmed cell death, significantly reducing tumor growth. Since targaprimir-96 was highly selective in its targeting, healthy cells were unaffected.

In contrast, Disney noted, a typical cancer therapeutic targets and kills cells indiscriminately, often leading to side effects that can make these drugs difficult for patients to tolerate.

Benjamin Zealley and Aubrey D.N.J. de Grey
Commentary on Some Recent Theses Relevant to Combating Aging: June 2015

REJUVENATION RESEARCH 2015; 18(3), 282 – 287

Cancer Autoantibody Biomarker Discovery and Validation Using Nucleic Acid Programmable Protein Array
Jie Wang, PhD, Arizona State University

Currently in the United States, many patients with cancer do not benefit from population-based screening due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and are diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies are a promising class of molecules that have the potential to serve as early detection biomarkers for cancers. This PhD thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancers, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformations on a protein array. This method adopted a novel protein tag technology, called a HaloTag, to immobilize proteins covalently on the surface of a glass slide. The covalent attachment allowed these proteins to endure harsh treatment without becoming dissociated from the slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to increase significantly the signal-to-noise ratio of protein array–based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases, 145 controls, 70 non-basal breast cancer) by enzyme-linked immunosorbent assay (ELISA), a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. A similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed tomography–positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA levels in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights into the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.

Comment: There are two widely supported models for cancer development and progression—the clonal evolution (CE) model and the cancer stem cell (CSC) model. Briefly, the former claims that most or all cells in a tumor contribute to its maintenance; as newer and more aggressive clones develop by random mutation, they become responsible for driving growth. The range of different mutational profiles generated is assumed to be large enough to account for disease recurrence after therapy (due to rare resistant clones) and metastasis (clones arising with the ability to travel to distant sites). The CSC model instead asserts that a small number of mutated stem cells are the origin of the primary cell mass, drive metastasis through the intermittent release of undifferentiated, highly mobile progeny, and account for recurrence due to a generally quiescent metabolic profile conferring potent resistance to chemotherapy. In either case, the immunological visibility of an early tumor may be highly sporadic. Clones arising early in CE differ little in proteomic terms from healthy host cells; those that do trigger a response are unlikely to have acquired robust resistance to immune attack, so are destroyed quickly in favor of their stealthier brethren. Likewise, CSCs share some of the immune privilege of normal stem cells and, due to their inherent ability to produce differentiated progeny with distinct proteomic signatures, are partially protected from attacks on their descendants. Consequently, such well-hidden cells may remain in the body for years to decades. The autoantibody panel developed in this study for basal-like breast cancer exhibits exceptional specificity despite a comparatively small training set. Given its ease of application, this suggests great promise for a more exhaustively trained classifier as a populationlevel screening tool.


Condition-Specific Differential Sub-Network Analysis for Biological Systems
Deepali Jhamb, PhD, Indiana University

Biological systems behave differently under different conditions. Advances in sequencing technology over the last decade have led to the generation of enormous amounts of condition-specific data. However, these measurements often fail to identify low-abundance genes and proteins that can be biologically crucial. In this work, a novel textmining system was first developed to extract condition-specific proteins from the biomedical literature. The literaturederived data was then combined with proteomics data to construct condition-specific protein interaction networks. Furthermore, an innovative condition-specific differential analysis approach was designed to identify key differences, in the form of sub-networks, between any two given biological systems. The framework developed here was implemented to understand the differences between limb regenerationcompetent Ambystoma mexicanum and regeneration-deficient Xenopus laevis. This study provides an exhaustive systems-level analysis to compare regeneration competent and deficient sub-networks to show how different molecular entities inter-connect with each other and are rewired during the formation of an accumulation blastema in regenerating axolotl limbs. This study also demonstrates the importance of literature-derived knowledge, specific to limb regeneration, to augment the systems biology analysis. Our findings show that although the proteins might be common between the two given biological conditions, they can have a high dissimilarity based on their biological and topological properties in the sub-network. The knowledge gained from the distinguishing features of limb regeneration in amphibians can be used in future to induce regeneration chemically in mammalian systems. The approach developed in this dissertation is scalable and adaptable to understanding differential sub-networks between any two biological systems. This methodology will not only facilitate the understanding of biological processes and molecular functions that govern a given system, but will also provide novel intuitions about the pathophysiology of diseases/conditions.

Comment: We have long advocated a principle of directly comparing young and old bodies as a means to identify the classes of physical damage that accumulate in the body during aging. This approach circumvents our ignorance of the full etiology of each particular disease manifestation, a phenomenally difficult question given the ethical issues of experimenting on human subjects, the lengthy ‘‘incubation time’’ of aging-related diseases, and the complex interconnections between their risk factors—innate and environmental. Repairing such damage has the potential to prevent pathology before symptoms appear, an approach now becoming increasingly mainstream.11 However, a naı¨ve comparison faces a number of difficulties, even given a sufficiently large sample set to compensate for inter-individual variation. Most importantly, the causal significance of a given species cannot be reliably determined from its simple prevalence.12 The catalytic nature of cell biology means that those entities whose abundance changes the most profoundly in absolute terms are quite unlikely to be the drivers of that change and may even spontaneously revert to baseline levels in the absence of on-going stimulation. Meanwhile, functionality is often heavily influenced independently of abundance by post-translational modifications that may escape direct detection. Sub-network analysis uses computational means to identify groups of genes and/or proteins that vary in a synchronized way with some parameter, indicating functional connectivity. The application of methods such as those developed here to the comparison of a wide range of younger and older conditions will facilitate the identification of processes—not merely individual factors—that are impaired with age, and thus will help greatly in identifying the optimal points for intervention.


Development of a Light Actuated Drug Delivery-on-Demand System
Chase Linsley, PhD, University of California, Los Angeles

The need for temporal–spatial control over the release of biologically active molecules has motivated efforts to engineer novel drug delivery-on-demand strategies actuated via light irradiation. Many systems, however, have been limited to in vitro proof-of-concept due to biocompatibility issues with the photo-responsive moieties or the light wavelength, intensity, and duration. To overcome these limitations, the objective of this dissertation was to design a light-actuated drug delivery-on-demand strategy that uses biocompatible chromophores and safe wavelengths of light, thereby advancing the clinical prospects of light-actuated drug delivery-on-demand systems. This was achieved by: (1) Characterizing the photothermal response of biocompatible visible light and near-infrared-responsive chromophores and demonstrating the feasibility and functionality of the light actuated on-demand drug delivery system in vitro; and (2) designing a modular drug delivery-on-demand system that could control the release of biologically active molecules over an extended period of time. Three biocompatible chromophores—Cardiogreen, Methylene Blue, and riboflavin—were identified and demonstrated significant photothermal response upon exposure to near-infrared and visible light, and the amount of temperature change was dependent upon light intensity, wavelength, as well as chromophore concentration. As a proof-of-concept, pulsatile release of a model protein from a thermally responsive delivery vehicle fabricated from poly(N-isopropylacrylamide) was achieved over 4 days by loading the delivery vehicle with Cardiogreen and irradiating with near-infrared light. To extend the useful lifetime of the light-actuated drug delivery-on-demand system, a modular, reservoir-valve system was designed. Using poly(ethylene glycol) as a reservoir for model small molecule drugs combined with a poly(N-isopropylacrylamide) valve spiked with chromophore-loaded liposomes, pulsatile release was achieved over 7 days upon light irradiation. Ultimately, this drug delivery strategy has potential for clinical applications that require explicit control over the presentation of biologically active molecules. Further research into the design and fabrication of novel biocompatible thermally responsive delivery vehicles will aid in the advancement of the light-actuated drug delivery-on-demand strategy described here. Comment: Our combined comments on this thesis and the next one appear after the next abstract.


Light-Inducible Gene Regulation in Mammalian Cells
Lauren Toth, PhD, Duke University

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue-engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible. The recent emergence of the field of optogenetics—the ability to control gene expression using light—has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific 3-bp DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or enhanced green fluorescent protein (eGFP) transgene enabled expression of the transgene in response to blue light. Gene activation was both reversible and tunable on the basis of duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be patterned spatially by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore, we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a lightactivated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the amino-terminal fragment of CIB1 to the amino, carboxyl, or amino and carboxyl terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic ‘‘on’’ switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase–mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system, could be used to express MyoD or the synthetic transcription factor VP64- MyoD in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated up-regulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T1/2 cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased micro-vessel density compared to mice that were not illuminated. Analysis of human vascular endothelial growth factor (VEGF) and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Comment: Although it is easy to characterize technological progress as following in the wake of scientific discoveries, the reverse is almost equally true; advances in technique open the door to types of experiment previously intractable or impossible. Such is currently the case for the field of optically controlled biotechnology, which has exploded into prominence, particularly over the last half-decade. Light of an appropriate wavelength can penetrate mammalian tissues to a depth of up to a couple of centimeters, rendering much of the living body accessible to optical study and control—still more if the detector/source is integrated into an endoscopic or fiber optic probe. Techniques borrowed from the semiconductor industry allow patterns of illumination to be controlled down to the nanometer scale, ideal for addressing individual cells. The highly controlled time course of such experiments, as compared to traditional means of gene activation, such as the addition of a chemical agent to the medium, eliminates confounding variables, and simplifies data analysis. Furthermore, this level of immediate control opens the door to closed-loop systems where the activity of entities under optical control can be continuously tuned in relation to some parameter(s). In the first of these two illuminating theses, a vehicle is developed that permits light-driven release of a small molecule. Such a system could be employed to target a systemically administered antibiotic or anti-neoplastic agent to a site of infection or cancer while sparing other bodily tissues from toxicity. Because most modern drugs cannot be produced in the body, even given arbitrarily good control of cellular biochemistry, this technique will have lasting value in numerous clinical contexts. In the second thesis, the level of precision achieved is even more profound; the CRISPR/Cas9 system has received much recent attention13 in its own right for its capacity to target arbitrary genetic sequences without an arduous protein-engineering step. The LACE system described stands to permit genetic manipulation with almost arbitrarily good spatial, temporal, and genomic site-specific control, using only means available to a typical university laboratory.


Targeting T Cells for the Immune-Modulation of Human Diseases
Regina Lin, PhD, Duke University

Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers, and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity, and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell–mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and pre-clinically characterizing their mechanisms of action and in vivo therapeutic efficacy.

CD8+ cytotoxic T cells have potent anti-tumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo– expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA (miRNA)-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR- 23a was up-regulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired anti-tumor potential of patient CD8+ T cells. We determined that tumor-derived transforming growth factor-b (TGF-b) directly suppresses CD8+ T cell immune function by elevating miR-23a and down-regulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-bmediated tumor immune-evasion pathway

Having established that miR-23a-inhibition can enhance the quality and functional resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers—such as TGF-b—within the tumor microenvironment. Because TGF-b poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wild-type and miR-23a–deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumorspecific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wild-type EGFRvIIICAR T cells were vulnerable to functional suppression by TGF-b, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF-b. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23adeficient EGFRvIII-CAR T cells for GBM immunotherapy.

Last, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immunesuppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro. Subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory interferon-c (IFN-c) and interleukin-17 (IL-17) production by fully differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.

Comment: Immunotherapy is among the most promising approaches to cancer treatment, having the specificity and scope to selectively target transformed cells wherever they may reside within the body and the potential to install a permanent defense against disease recurrence. By the time a typical cancer is clinically diagnosed, however, it has already found means to survive a prolonged period of potential immune attack. The mechanisms by which tumors evade immune surveillance are beginning to be elucidated,15,16 and include both direct suppression of effector cells and progressive editing of the host’s immune repertoire to disfavor future attack. It is inherently difficult to interfere with these defenses directly, due to the selection pressures in genetically heterogeneous neoplastic tissue. Much effort is thus being focused on methods for rendering therapeutically delivered immune cells resistant to their effects. The cytokine TGF-b is paradoxically known to function as both a tumor suppressor in healthy tissue and as a tumorderived species associated with multiple cancer-promoting activities, including enhanced immune evasion. This work identifies the pathway by which TGF-b compromises cytotoxic T cell function in the tumor microenvironment, and demonstrates an effective method for blocking this signal. In many clinical cases, however, editing of the patient’s immune repertoire has already removed or rendered anergic those immune cells able to recognize their cancer. Thus, the finding that blocking TGF-b signaling also appears to enhance the effectiveness of CAR-modified T cells— engineered with an antibody fragment targeting them with high affinity to a particular tumor-associated epitope—is a welcome addition to these already promising results.


Novel Fibonacci and non-Fibonacci structure in the sunflower: results of a citizen science experiment

Jonathan Swinton, Erinma Ochu, The MSI Turing’s Sunflower Consortium

Published 18 May 2016. DOI

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

  1. Introduction

Fibonacci structure can be found in hundreds of different species of plants [1]. This has led to a variety of competing conceptual and mathematical models that have been developed to explain this phenomenon. It is not the purpose of this paper to survey these: reviews can be found in [14], with more recent work including [510]. Instead, we focus on providing empirical data useful for differentiating them.

These models are in some ways now very mathematically satisfying in that they can explain high Fibonacci numbers based on a small number of plausible assumptions, though they are not so satisfying to experimental scientists [11]. Despite an increasingly detailed molecular and biophysical understanding of plant organ positioning [1214], the very parsimony and generality of the mathematical explanations make the generation and testing of experimental hypotheses difficult. There remains debate about the appropriate choice of mathematical models, and whether they need to be central to our understanding of the molecular developmental biology of the plant. While sunflowers provide easily the largest Fibonacci numbers in phyllotaxis, and thus, one might expect, some of the stronger constraints on any theory, there is a surprising lack of systematic data to support the debate. There have been only two large empirical studies of spirals in the capitulum, or head, of the sunflower: Weisse [15] and Schoute [16], which together counted 459 heads; Schoute found numbers from the main Fibonacci sequence 82% of the time and Weise 95%. The original motivation of this study was to add a third replication to these two historical studies of a widely discussed phenomenon. Much more recently, a study of a smaller sample of 21 seedheads was carried out by Couder [17], who specifically searched for non-Fibonacci examples, whereas Ryan et al. [18] studied the arrangement of seeds more closely in a small sample of Helianthus annuus and a sample of 33 of the related perennial H. tuberosus.

Neither the occurrence of Fibonacci structure nor the developmental biology leading to it are at all unique to sunflowers. As common in other species, the previous sunflower studies found not only Fibonacci numbers, but also the occasional occurrence of the double Fibonacci numbers, Lucas numbers and F4 numbers defined below [1]. It is worth pointing out the warning of Cooke [19] that numbers from these sequences make up all but three of the first 17 integers. This means that it is particularly valuable to look at specimens with large parastichy numbers, such as the sunflowers, where the prevalence of Fibonacci structure is at its most striking.

Neither Schoute nor Weisse reported their precise technique for assigning parastichy numbers to their samples, and it is noteworthy that neither author reported any observation of non-Fibonacci structure. One of the objectives of this study was to rigorously define Fibonacci structure in advance and to ensure that the assignment method, though inevitably subjective, was carefully documented.

This paper concentrates on the patterning of seeds towards the outer rim of sunflower seedheads. The number of ray florets (the ‘petals’, typically bright yellow) or the green bracts behind them tends to have a looser distribution around a Fibonacci number. In the only mass survey of these, Majumder & Chakravarti [20] counted ray florets on 1002 sunflower heads and found a distribution centred on 21.

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

Incorporation of irregularity into the mathematical models of phyllotaxis is relatively recent: [17] gave an example of a disordered pattern arising directly from the deterministic model while more recently the authors have begun to consider the effects of stochasticity [10,21]. Differentiating between these models will require data that go beyond capturing the relative prevalence of different types of Fibonacci structure, so this study was also designed to yield the first large-scale sample of disorder in the head of the sunflower.

The Fibonacci sequence is the sequence of integers 1,2,3,5,8,13,21,34,55,89,144… in which each member after the second is the sum of the two preceding. The Lucas sequence is the sequence of integers 1,3,4,7,11,18,29,47,76,123… obeying the same rule but with a different starting condition; the F4 sequence is similarly 1,4,5,9,14,23,37,60,97,…. The double Fibonacci sequence 2,4,6,10,16,26,42,68,110,… is double the Fibonacci sequence. We say that a parastichy number which is any of these numbers has Fibonacci structure. The sequencesF5=1,5,6,11,17,28,45,73,… and F8=1,8,9,17,26,43,69,112… also arise from the same rule, but as they had not been previously observed in sunflowers we did not include these in the pre-planned definition of Fibonacci structure for parsimony. One example of adjacent pairs from each of these sequences was, in fact, observed but both examples are classified as non-Fibonacci below. A parastichy number which is any of 12,20,33,54,88,143 is also not classed as having Fibonacci structure but is distinguished as a Fibonacci number minus one in some of the analyses, and similarly 14,22,35,56,90,145 as Fibonacci plus one.

When looking at a seedhead such as in figure 1 the eye naturally picks out at least one family of parastichies or spirals: in this case, there is a clockwise family highlighted in blue in the image on the right-hand side.

Distribution and type of parastichy pairs

Figure 5 plots the individual pairs observed. On the reference line, the ratio of the numbers is equal to the golden ratio so departures from the line mark departures from Fibonacci structure, which are less evident in the more reliable photoreviewed dataset. It can be seen from table 3 that Fibonacci pairings dominate the dataset.

Table 3.

Observed pairings of Fibonacci types of clockwise and anticlockwise parastichy numbers. Other means any parastichy number which neither has Fibonacci structure nor is Fibonacci ±1. Of all the Fibonacci ±1/Fibonacci pairs, only sample 191, a (21,20) pair, was not close to an adjacent Fibonacci pair.

One typical example of a Fibonacci pair is shown in figure 6, with a double Fibonacci case infigure 1 and a Lucas one in figure 7. There was no photoreviewed example of an F4 pairing. The sole photoreviewed assignment of a parastichy number to the F4 sequence was the anticlockwise parastichy number 37 in sample 570, which was relatively disordered. The clockwise parastichy number was 55, lending support to the idea this may have been a perturbation of a (34,55) pattern. We also found adjacent members of higher-order Fibonacci series. Figures 8 and 9 each show well-ordered examples with parastichy counts found adjacent in the F5 and F8 series, respectively: neither of these have been previously reported in the sunflower.

Figure 6.

Sunflower 095. An (89,55) example with 89 clockwise parastichies and 55 anticlockwise ones, extending right to the rim of the head. Because these are clear and unambiguous, the other parastichy families which are visible towards the centre are not counted here.

Figure 7.   Sunflower 171. A Lucas series (76,47) example.

Sunflower 667. Anticlockwise parastichies only, showing competing parastichy families which are distinct but in some places overlapping.

Our core results are twofold. First, and unsurprisingly, Fibonacci numbers, and Fibonacci structure more generally, are commonly found in the patterns in the seedheads of sunflowers. Given the extent to which Fibonacci patterns have attracted pseudo-scientific attention [33], this substantial replication of limited previous studies needs no apology. We have also published, for the first time, examples of seedheads related to the F5 and F8 sequences but by themselves they do not add much to the evidence base. Our second core result, though, is a systematic survey of cases where Fibonacci structure, defined strictly or loosely, did not appear. Although not common, such cases do exist and should shed light on the underlying developmental mechanisms. This paper does not attempt to shed that light, but we highlight the observations that any convincing model should explain. First, the prevalence of Lucas numbers is higher than those of double Fibonacci numbers in all three large datasets in the literature, including ours, and there are sporadic appearances of F4, F5 and F8 sequences. Second, counts near to but not exactly equal to Fibonacci structure are also observable: we saw a parastichy count of 54 more often than the most common Lucas count of 47. Sometimes, ambiguity arises in the counting process as to whether an exact Fibonacci-structured number might be obtained instead, but there are sufficiently many unambiguous cases to be confident this is a genuine phenomenon. Third, among these approximately Fibonacci counts, those which are a Fibonacci number minus one are significantly more likely to be seen than a Fibonacci number plus one. Fourth, it is not uncommon for the parastichy families in a seedhead to have strong departures from rotational symmetry: this can have the effect of yielding parastichy numbers which have large departures from Fibonacci structure or which are completely uncountable. This is related to the appearance of competing parastichy families. Fifth, it is common for the parastichy count in one direction to be more orderly and less ambiguous than that in the other. Sixth, seedheads sometimes possess completely disordered regions which make the assignment of parastichy numbers impossible. Some of these observations are unsurprising, some can be challenged by different counting protocols, and some are likely to be easily explained by the mathematical properties of deformed lattices, but taken together they pose a challenge for further research.

It is in the nature of this crowd-sourced experiment with multiple data sources that it is much easier to show variability than it is to find correlates of that variability. We tried a number of cofactor analyses that found no significant effect of geography, growing conditions or seed type but if they do influence Fibonacci structure, they are likely to be much easier to detect in a single-experimenter setting.

We have been forced by our results to extend classifications of seedhead patterns beyond structured Fibonacci to approximate Fibonacci ones. Clearly, the more loose the definition of approximate Fibonacci, the easier it is to explain away departures from model predictions. Couder [17] found one case of a (54,87) pair that he interpreted as a triple Lucas pair 3×(18,29). While mathematically true, in the light of our data, it might be more compellingly be thought of as close to a (55,89) ideal than an exact triple Lucas one. Taken together, this need to accommodate non-exact patterns, the dominance of one less over one more than Fibonacci numbers, and the observation of overlapping parastichy families suggest that models that explicitly represent noisy developmental processes may be both necessary and testable for a full understanding of this fascinating phenomenon. In conclusion, this paper provides a testbed against which a new generation of mathematical models can and should be built.


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