New process developed to activate genes on demand




When it comes to gene expression – the process by which our DNA provides the recipe used to direct the synthesis of proteins and other molecules that we need for development and survival – scientists have so far studied one single gene at a time.

However, a new approach developed by Harvard geneticist George Church, Ph.D., can help uncover how tandem gene circuits dictate life processes, such as the healthy development of tissue or the triggering of a particular disease, and can also be used for directing precision stem cell differentiation for regenerative medicine and growing organ transplants.

The findings, reported by Church and his team of researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School in , show promise that precision gene therapies could be developed to prevent and treat disease on a highly customizable, personalized level, which is crucial given that diseases develop among diverse pathways among genetically varied individuals.

The approach leverages the Cas9 protein, which has already been employed as a Swiss Army knife for genome engineering, in a novel way. The Cas9 protein can be programmed to bind and cleave any desired section of DNA, but now Church’s new approach activates the genes, and binds rather than cleaving them, triggering them to activate transcription to express or repress desired genetic traits.

By engineering the Cas9 to be fused to a triple-pronged transcription factor, Church and his team can robustly manipulate single or multiple genes to control gene expression. “In terms of genetic engineering, the more knobs you can twist to exert control over the expression of genetic traits, the better,” said Church, a Wyss Core Faculty member who is also Professor of Genetics at Harvard Medical School and Professor of Health Sciences and Technology at Harvard and MIT. “We could essentially dial gene expression up or down with great precision.”

Such a capability could lead to gene therapies that would mitigate age-related degeneration and the onset of disease. In the study, Church and his team demonstrated the ability to manipulate gene expression in yeast, flies, mouse and human cell cultures. “We envision using this approach to investigate and create comprehensive libraries that document which gene circuits control a wide range of gene expression,” said one of the study’s lead authors Alejandro Chavez, Ph.D., Postdoctoral Fellow at the Wyss Institute.
The new Cas9 approach could also potentially target and activate sections of the genome made up of genes that are not directly responsible for transcription. These sections, which comprise up to 90% of the genome in humans, have previously been considered to be useless DNA “dark matter” by geneticists. In contrast to translated DNA, which contains recipes of genetic information used to express traits, this DNA dark matter contains transcribed genes which act in mysterious ways, with several of these genes often having influence in tandem.

But now, that DNA dark matter could be accessed, allowing scientists to document which non-translated genes can be activated in tandem to influence gene expression. These non-translated genes could also be turned into a docking station of sorts.

To demonstrate this point, the researchers used it to grow brain neuron cells from stem cells and found that using the approach to program development of neuronal cells was 40–fold more successful than prior established methods. This is the first time that Cas9 has been leveraged to efficiently differentiate stem cells into brain cells, and can also be used in combination with other gene editing technologies.

UK scientists create cartilage from stem cells

Scientists have succeeded in producing cartilage formed from embryonic stem cells that could in future be used to treat osteoarthritis.

In research funded by Arthritis Research UK, Professor Sue Kimber and her team in the Faculty of Life Sciences at the University of Manchester have developed a protocol under strict laboratory conditions to grow and transform embryonic stem cells into cartilage cells (also known as chondrocytes).

“This work represents an important step forward in treating cartilage damage by using embryonic stem cells to form new tissue, although it’s still in its early experimental stages,” said Kimber. Their research was published in Stem Cells Translational Medicine.

During the study, the team analysed the ability of embryonic stems cells to become precursor cartilage cells. They were then implanted into cartilage defects in the knee joints of rats. After four weeks, cartilage was partially repaired and following 12 weeks, a smooth surface, which appeared similar to normal cartilage, was observed.

Further study of this newly regenerated cartilage showed that cartilage cells from embryonic stem cells were still present and active within the tissue. Developing and testing this protocol in rats is the first step in generating the information needed to run a study in people with arthritis. However, more data will need to be collected to check that this protocol is effective and that there are no toxic side-effects.

Chondrocytes created from adult stem cells are currently being experimentally used, but as they cannot yet be produced in large amounts, the procedure is expensive. With their huge capacity to proliferate, embryonic stem cells, which can be manipulated to form almost any type of mature cell, offer the possibility of highvolume production of cartilage cells.

Their use would also be cheaper and applicable to greater number of arthritis patients, the researchers claim. Osteoarthritis affects more than eight million people in the UK, and is a major cause of disability. It occurs when cartilage at the ends of bones wears away causing joint pain and stiffness.

“Current treatments of osteoarthritis are restricted to relieving painful symptoms, with no effective therapies to delay or reverse cartilage degeneration. Joint replacements are successful in older patients but not young people, or athletes who’ve suffered a sports injury,” added Director of Research at Arthritis Research UK, Dr Stephen Simpson, added.


Nanoparticles could impact medication compliance in cornea transplants

Johns Hopkins Medicine researchers may have discovered a way to prevent rejection by using biodegradable nanoparticles that release needed medication into the eye after surgery. This discovery could solve the decades-old issue of medication compliance and help patients achieve corneal transplant success.

About 48,000 corneal transplants are done each year in the US, compared to approximately 16,000 kidney transplants and 2,100 heart transplants. Of the 48,000 corneal transplants, 10% of them end up in rejection, largely due to poor medication compliance. This costs the health care system and puts undue strain on clinicians, patients and their families.

“About 60 to 80% of patients don’t take medicine the way they are supposed to,” says Walter Stark, M.D., chief of the Division of Cornea, Cataract and External Eye Diseases at Johns Hopkins. In an animal study published in Journal of Controlled Release, researchers looked into ways to alleviate the strain of adhering to a post-surgery treatment regimen that is sometimes hard to manage.

Rats that underwent a corneal graft surgery were randomly divided into four groups and were given various treatments. One group was injected weekly for nine weeks with a safe, biodegradable nanoparticle loaded with corticosteroids for timed release of medicine.

The other three groups received weekly injections of saline, placebo nanoparticles and free dexamethasone sodium phosphate aqueous solution after surgery, respectively. Treatments were given until the graft was clinically deemed as failed or until the nine-week test period concluded. Researchers looked at corneal transparency, swelling and growth of new blood vessels to decide if a graft had failed.

For rats that received the nanoparticle loaded with corticosteroids, 65% of the treatment remained in the eye and did not leak within one week of the surgery. The concentration of the treatment also remained stronger than in the other three treatment groups.

Additionally, there were no signs of swelling, and the cornea was clear throughout the test period. There were also far fewer instances of unwanted growth of new blood vessels in this group.

Two weeks after surgery, rats that received the placebo nanoparticle and saline injections had severe swelling, opaque corneas and unwanted growth of new blood vessels, all indicating graft failure. After four weeks, rats that received free dexamethasone sodium phosphate aqueous solution all had graft failure as well. The only group that showed successful corneal transplant was the group of rats that received the corticosteroid-loaded nanoparticle injections.

The grafts were still viable in 100% of these rats. The steroid-loaded nanoparticle treatment group showed no signs of corneal transplant rejection. “That’s 100% efficacy, a very promising finding,” says Justin Hanes, Ph.D., director of the Center for Nanomedicine. “This type of treatment may also help prevent corneal transplant rejection in humans while making medicine adherence much easier on patients and their families.” The nanoparticle loaded with medication could eliminate the need for a patient to remember to take their medicine ? often multiple doses per hour ? after a surgery, alleviating compliance risk.

These types of drug delivery systems could be paired with other drugs and used in other conditions, such as glaucoma, macular degeneration and corneal ulcers, among others. Funding of this study came from the Raymond Kwok Family Research Fund, a grant from the King Khaled Eye Specialist Hospital of Saudi Arabia and the Eye Bank Association of America/Richard Lindstrom Research Grant 2012.


Cardiovascular risks among obese adolescents assessed

Cardiovascular risks of severe paediatric obesity, assessed among adolescents participating in the “Teen Longitudinal Assessment of Bariatric Surgery” (Teen-LABS) study, were published this week in JAMA Pediatrics. Teen-LABS is a multi-centre clinical study funded by the National Institute of Diabetics and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH) that is examining the safety and health effects of surgical weight loss procedures.

“This NIH-funded study will add important knowledge to the field of severe obesity during adolescence and the effects of bariatric surgery,” said Dr Marc P. Michalsky, MD, FACS, FAAP, surgical director of the Centre for Healthy Weight and Nutrition at Nationwide Children’s and Associate Professor of Clinical Surgery and Pediatrics at The Ohio State University College of Medicine. “Collaborating with colleagues around the country in a study of this magnitude to gather critical data defining cardiovascular disease (CVD) and other health risks, is both gratifying and hugely important. The results of this study will improve our understanding of the significant medical challenges faced by severely obese teens as well as document outcomes following surgical weight loss.”

In this most recent publication from the Teen-LABS research study, investigators note that while paediatric obesity is more common now than in previous decades, very little is known about the CVD risks in the most severely obese teens. The main goal of the current publication was to assess the baseline prevalence and predictors of CVD risks among severely obese adolescents before undergoing weight-loss surgery.

The authors of this publication found that severely obese adolescents carry not only excess weight, but also have much higher risk for CVD than previously realized. Of the 242 participants in the Teen-LABS cohort, 95% had at least one CVD risk factor. Seventy-five percent had elevated blood pressure (including hypertension and pre-hypertension), 50% had unhealthy cholesterol levels, and nearly three-quarters of the group were insulin resistant. Importantly, the study also confirmed that increasing weight in teenagers is associated with increases in blood sugar and blood pressure.

While the majority of study participants are female, researchers found an interesting link between gender and CVD. “We found that adolescent boys were at a markedly higher risk compared with adolescent girls for abnormal triglyceride levels,” said Dr Michalsky. “Among severely obese adolescents, recognition and treatment of CVD risk factors is important to help limit further progression of disease.”


URI researchers invent lab-on-paper for rapid, inexpensive medical diagnostics



A team of University of Rhode Island (URI) engineers led by Professor Mohammad Faghri has created a new paper-based platform for conducting a wide range of complex medical diagnostics. The key development was the invention of fluid-actuated valves embedded in the paper that allow for sequential manipulation of sample fluids and multiple reagents in a controlled manner to perform complex multi-step immune-detection tests without human intervention.

Faghri said the platform technology can be potentially applied to a wide variety of medical diagnostics, from Lyme disease and HIV to Ebola and malaria. “If someone comes up with a new biomarker for detecting a disease, we can create a test for it using our platform,” he said. He also envisions applications in the veterinary medicine field, as well as for the detection of environmental contaminants and biological or chemical threats. “It could even be used at airports to test fluids for possible bioterror agents,” he added.

A number of companies have already expressed interest in adapting various applications to the new platform.

A strong patent with broad claims has been issued by the US Patent and Trademark Office for this technology, and two more are pending. According to Faghri, paper-based lateral flow test strips, such as for pregnancy tests, have been commercially successful for many years. In these devices, a sample fluid wicks along a strip of paper, reacts with embedded reagents, and produces a coloured signal result.

However, more complex medical diagnostics such as enzymatic assay protocols require multiple reagents triggered at particular times during the process, which can only be accomplished autonomously using the proprietary microfluidic valve technology created by the URI research team.

“We combined the well-established test strip technology, micro-patterning techniques and our innovative paper-based valves to create a new class of strip tests capable of autonomously handling multiple reagents,” explained Faghri. “The sample fluid activates the flow of reagents in a predetermined sequence and time. When combined with an optical reader, which could even be a conventional smart phone, the lab-on-paper device provides accurate quantitative results.

“We’re the only research group in the world to have created fluidic valves on multilayered paper without the use of external mechanical, electric or magnetic force and to use these valves to create fluidic circuits similar to electrical circuits,” he added. The lab-on-paper devices are constructed with multiple layers of paper printed with wax to create a three-dimensional structure of valves and channels along which the fluid travels, triggering the reagents at the appropriate time and generating a result.

Faghri and collaborator Constantine Anagnostopoulos, a URI adjunct professor of mechanical engineering, established a startup company, Labonachip LLC, to commercialize their technologies. Anagnostopoulos serves as the company’s president. “Our next step is to find investors to help take us to the next level,” said Anagnostopoulos. The researchers have already succeeded in performing a feasibility study of their technology by detecting a biomarker for sepsis, a life-threatening complication from an infection.

ProThera Biologics, a Providence-based company co-founded by Brown University Professor Yow-Pin Lim, identified a biomarker that indicates a patient is going into shock from sepsis, and the company has collaborated with Faghri and Anagnostopoulos to develop a paperbased rapid test using this biomarker.

The researchers are working with the University of Rhode Island Research Foundation to identify potential partners. “A number of diagnostics companies are currently evaluating this lab-on-paper microfluidics technology as the industry moves from lab-based testing to point-of-care rapid, autonomous diagnostics,” said Gerald Sonnenfeld, chairman of the URI Research Foundation and URI’s vice president of research and economic development. “We expect the point-of-care testing market to expand greatly over the next several years.”


Scientists crack a piece of the neural code for learning and memory

In a work published in Nature, researchers at Cold Spring Harbor Laboratory (CSHL) describe how post-mortem brain slices can be “read” to determine how a rat was trained to behave in response to specific sounds before it died. The work provides one of the first examples of how changes in the activity of individual neurons encode learning and memory in the brain.

Researchers have long hypothesized that changes in neuronal activity are responsible for our ability to make decisions, remember things, and learn. “Neuroscientists have previously identified brain areas involved in learning something,” says CSHL Professor Anthony Zador, who led the team of researchers on this current work. “But we wanted to drill down further and identify how changes at specific connections encode a particular behavioral response.”

To do this, the team focused on how rats translate sound cues into behaviour. The researchers trained rats to associate a specific tone with a reward. Changes in the tone – like the difference between a tuba and a flute – signalled the animal to look for the reward either on the left or right side of a training box.

In previous work, the team discovered that activity in specific populations of neurons was crucial for animals to perform the task. This neuronal population transmitted information from one auditory brain region (the auditory cortex) to another (the auditory striatum).

In the current work, the team measured the strength of the connections between these two populations of neurons, as animals learned the task. “We found that there was a gradient in activity across the auditory striatum that corresponded to whether the animal was trained to go left or right for their reward,” explained Zador. Based upon this information, the team reasoned that they might be able to use post-mortem brain slices to “predict” (obviously, in retrospect) how these or other rats had been trained. As Zador describes, “We were amazed that in all cases, our predictions – left or right – were correct. We had deciphered a tiny piece of the neural code with which the animal encoded these memories. In essence, we could read the minds of these rats.”

According to Zador, the results are likely to be broadly applicable to other senses and parts of the brain. “We are excited to apply this method to more complex forms of learning, and to other sensory systems, like vision.” The paper, supported by grants from the US National Institutes of Health and the Swartz Foundation, can be obtained online at: http://dx.doi.org/10.1038/nature14225


Vitamin D deficiency linked more closely to diabetes than obesity

People who have low levels of vitamin D are more likely to have diabetes, regardless of how much they weigh, according to a new study published in the Endocrine Society’s Journal of Clinical Endocrinology & Metabolism.

The results help clarify the connection between vitamin D, obesity and diabetes. According to the Society’s Scientific Statement on the Noon-skeletal effects of vitamin D, studies have found that people who have low levels of vitamin D are more likely to be obese. They also are more likely to have Type 2 diabetes, pre-diabetes and metabolic syndrome than people with normal vitamin D levels. Vitamin D helps the body absorb calcium and maintain bone and muscle health.

The skin naturally produces this vitamin after exposure to sunlight. People also absorb smaller amounts of the vitamin through foods, such as milk fortified with vitamin D. More than one billion people worldwide are estimated to have deficient levels of vitamin D due to limited sunshine exposure.

“The major strength of this study is that it compares vitamin D levels in people at a wide range of weights (from lean to morbidly obese subjects) while taking whether they had diabetes into account,” said one of the study’s authors, Mercedes Clemente- Postigo, MSc, of Instituto de Investigacion Biomedica de Malaga (IBIMA) at Complejo Hospitalario de Malaga (Virgen de la Victoria) and Universidad de Malaga in Malaga, Spain.

The cross-sectional study compared vitamin D biomarkers in 118 participants at the university hospital Virgen de la Victoria in Malaga as well as 30 participants from the Hospital Universitari Dr. Josep Trueta in Girona, Spain.

All participants were classified by their body-mass index (BMI) as well as whether they had diabetes, pre-diabetes or no glycaemic disorders. Researchers measured levels of vitamin D in the participants’ blood streams and vitamin D receptor gene expression in adipose tissue. The analysis found that obese subjects who did not have glucose metabolism disorders had higher levels of vitamin D than diabetic subjects. Likewise, lean subjects with diabetes or another glucose metabolism disorder were more likely to have low levels of vitamin D. Vitamin D levels were directly correlated with glucose levels, but not with BMI.

“Our findings indicate that vitamin D is associated more closely with glucose metabolism than obesity,” said one of the study’s authors, Manuel Macias-Gonzalez, PhD, of Complejo Hospitalario de Malaga (Virgen de la Victoria) and the University of Malaga. “The study suggests that vitamin D deficiency and obesity interact synergistically to heighten the risk of diabetes and other metabolic disorders. The average person may be able to reduce their risk by maintaining a healthy diet and getting enough outdoor activity.”

The study, “Serum 25-Hydroxyvitamin D and Adipose Tissue Vitamin D Receptor Gene Expresion: Relationship with Obesity and Type 2 Diabetes,” was published online, ahead of print.


                                  
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