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 Churchs new approach activates the genes,
and binds rather than cleaving them, triggering them to
activate transcription to express or repress desired
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 studys 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
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 40fold 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
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
This work represents an important step forward in
treating cartilage damage by using embryonic stem cells
to form new tissue, although its 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
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 whove suffered a
sports injury, added Director of Research at
Arthritis Research UK, Dr Stephen Simpson, added.
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 dont 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
The grafts were still viable in 100% of these rats. The
steroid-loaded nanoparticle treatment group showed no
signs of corneal transplant rejection. Thats
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
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
Childrens 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
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
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
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.
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.
Were 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 companys 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 URIs vice president of research and
economic development. We expect the point-of-care
testing market to expand greatly over the next several
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:
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
Societys Journal of Clinical Endocrinology &
The results help clarify the connection between vitamin
D, obesity and diabetes. According to the Societys
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 studys 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,
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
Our findings indicate that vitamin D is associated
more closely with glucose metabolism than obesity,
said one of the studys 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.