Study Shows Why We Are Made of DNA and Not RNA


In Brief

The most important distinction between DNA and RNA is DNA’s ability to morph when its bases experience chemical damage. DNA can dynamically accommodate base pairs in either Watson-Crick or Hoogsteen configurations, RNA stiffens and falls apart.

Milestones in DNA

In 1869, Friedrich Miescher discovered DNA. About seventy years thereafter, Oswald Avery and his colleagues proved that DNA carries genetic information. A decade later, James Watson and Francis Crick described the structure of the DNA and published their model of the DNA double helix.

Although it has been more than a century since DNA was first identified, scientists continue to learn more about this complex molecule and why we regard it as “the source code of life itself.”

Now, research led by Hashim Al-Hashimi from Duke University provides the most recent milestone in DNA history, showing why DNA, and not RNA, forms the blueprint for life. But first, let us review the basic properties and differences between DNA and RNA.

Source: DifferenceBetween.net

Understanding DNA and RNA

DNA and RNA are both nucleic acids (long biological macromolecules consisting of smaller molecules called nucleotides).

DNA is a double-stranded molecule, with two nucleotide strands consisting of a phosphate backbone, deoxyribose sugar, and has adenine, guanine, cytosine, and thymine as bases; whereas RNA is a single-stranded helix consisting of shorter chains of nucleotides, a phosphate backbone, ribose sugar, and has adenine, guanine, cytosine, and uracil bases.

But perhaps the most important difference between the two, as is shown in Al-Hashimi’s study, is that DNA is capable of shapeshifting in a way that RNA can’t, making DNA a more resilient and a better repository of our genetic code. Specifically, it has to do with Hoogsteen base pairs, which is when one of the nucleic acids is flipped 180 degrees. Ultimately, this causes the whole double helix structure to bend or “kink,” meaning that DNA can accommodate damage by turning in this manner. RNA molecules are incapable of forming Hoogsteen pairs, and so they fall apart—completely unraveling when they encountered the methyl group that the team introduced.

Al-Hashimi tells The Washington Post, “if our genomes were made up of RNA, there’s a very good chance that they wouldn’t be able to sustain chemical damage that’s inflicted on them all the time. It seems like DNA’s ability to absorb damage is one reason why we evolved DNA-based genomes.”

There is still a lot left to discover about our DNA, but we are coming closer to understanding why it is so effective and resilient.

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New Theory on Origin of Life on Earth Questions ‘RNA World’ Hypothesis


DNA com GGN

A new study from The Scripps Research Institute (TSRI) has offered a surprising twist of how life began on earth, questioning the likeability of the “RNA World” Hypothesis and proposing that DNA may have also existed when life began.
A new study from The Scripps Research Institute (TSRI) has offered a surprising twist of how life began on earth, questioning the likeability of the “RNA World” Hypothesis and proposing that DNA may have also existed when life began.

According to the study published in the journal Angewandte Chemie International Edition, scientists from TSRI said that the RNA World Hypothesis, which states that proteins and DNA originated from RNA molecules, could not be entirely true. They suggested that something could have existed along RNA to help it evolve.

“Why not think of RNA and DNA rising together, rather than trying to convert RNA to DNA by means of some fantastic chemistry at a prebiotic stage?” said Ramanarayanan Krishnamurthy, associate professor of chemistry at TSRI and senior author of the new study, via Science Daily.

To recall, scientists have been studying the RNA World Hypothesis for years. The hypothesis claims that self-replicating RNA, which came from multiple chemical reactions, led to the evolution of proteins and enzymes. These byproducts of RNA mark the birth of life on Earth.

At first, scientists thought that DNA and RNA could have merged together to create “heterogeneous” strands that would result to blended “chimeras.” However, upon testing, the scientists found that the mixed-up RNA and DNA strands are unstable, especially when the two share the same backbone.

Thus, the researchers came up with an alternate theory, saying that instead of creating “chimeras,” RNA and DNA could have evolved at the same time.

This means that apart from RNA, DNA could have also evolved separately in its own homogeneous system. The theory of RNA producing DNA, according to the researchers, could still be possible but it could have occurred after RNA met DNA.

Watch the vido discussion. URL:https://youtu.be/K1xnYFCZ9Yg

Scientists say the ‘R’ in RNA may be abundant in space


Scientists say the 'R' in RNA may be abundant in space
Ribose and a diversity of structurally related sugar molecules are formed by a formose-type reaction in evolved pre-cometary ice analogues and detected by multidimensional gas chromatography. Ribose sugars make up the backbone of ribonucleic acid (RNA), which is what scientists think coded the genetic instructions for living things before the emergence of DNA.

New research suggests that the sugar ribose – the “R” in RNA – is probably found in comets and asteroids that zip through the solar system and may be more abundant throughout the universe than was previously thought.

The finding has implications not just for the study of the origins of life on Earth, but also for understanding how much life there might be beyond our planet.

Scientists already knew that several of the molecules necessary for life including , nucleobases and others can be made from the interaction of cometary ices and space radiation. But ribose, which makes up the backbone of the RNA molecule, had been elusive – until now.

The new work, published Thursday in Science, fills in another piece of the puzzle, said Andrew Mattioda, an astrochemist at NASA Ames Research Center, who was not involved with the study.

“If all these molecules that are necessary for life are everywhere out in space, the case gets a lot better that you’ll find life outside of Earth,” he said.

RNA, which stands for ribonucleic acid, is one of the three macromolecules that are necessary for all life on Earth – the other two are DNA and proteins.

Many scientists believe that RNA is a more ancient molecule than DNA and that before DNA came on the scene, an “RNA world” existed on Earth. However, ribose, a key component in RNA, only forms under specific conditions, and scientists say those conditions were not present on our planet before life evolved. So, where did the ribose in the first RNA strands come from?

Scientists say the 'R' in RNA may be abundant in space
Ribose – a key molecule for the origin of life – detected in an interstellar ice analogue using multidimensional gas chromatography. Ribose sugars make up the backbone of ribonucleic acid (RNA) molecule, which is involved in protein synthesis in living cells. Credit: C. Meinert, CNRS

To see if these molecules could have been delivered to Earth by asteroids and comets, a team of researchers re-created the conditions of the early solar system in a French lab to see whether ribose could easily be made in space.

They started with water, methanol and ammonia because these molecules were abundant in the protoplanetary disk that formed around the sun at the dawn of the solar system, and are also abundant in gas clouds throughout the universe. They were put in a vacuum and then cooled to a cryogenic temperature of 80 degrees kelvin (minus-328 degrees Fahrenheit).

The resulting ices were then heated to room temperature, which caused the volatile molecules to sublimate, leaving a thin film of material.

“The simulation is of cometary ices only, not cometary dust grains,” said Uwe Meierhenrich, a chemist at the University of Nice Sophia Antipolis in France and one of the authors of the study.

The experiment took about six days to complete and yielded just 100 micrograms of the artificial cometary ice residue in the lab.

Artificial cometary ices have been created hundreds of times before in labs around the world, but until now researchers have not had the tools to detect sugars such as ribose in the samples.

Cornelia Meinert, also of the University of Nice Sophia Antipolis, explained that it’s not just sugar and sugar-related molecules that are created in these experiments, but also amino acids, carboxylic acids and alcohols.

“We are confronted with a very complex sample containing a huge diversity of molecules,” she said. “The identification of individual compounds is therefore very difficult.”

Meinert said it wasn’t until the group was able to use a new technique called multidimensional gas chromatography that they were able to detect ribose in these samples at all.

The researchers say that the ice samples they made in the lab could easily be made in other parts of the .

“Our ice simulation is a very general process that can occur in molecular clouds as well as in protoplanetary disks,” Meinert said. “It shows that the of the potentially first genetic material are abundant in interstellar environments.”

Scott Sandford, an astrochemist who has done similar work with cometary ices at NASA Ames Research Center, said adding sugars to the list of that can be forged in space is an important step in understanding what building blocks of life may be available to foster life in other worlds.

“Insofar as these materials play a role in getting started on planets, the odds are good that they’ll be present to help,” he said.

 

RNA combination therapy for lung cancer offers promise for personalized medicine.


Small RNA molecules, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), offer tremendous potential as new therapeutic agents to inhibit cancer-cell growth. However, delivering these small RNAs to solid tumors remains a significant challenge, as the RNAs must target the correct cells and avoid being broken down by enzymes in the body. To date, most work in this area has focused on delivery to the liver, where targeting is relatively straightforward.

This week in the journal Proceedings of the National Academy of Sciences, researchers at the Koch Institute for Integrative Cancer Research at MIT report that they have successfully delivered small RNA therapies in a clinically relevant of to slow and shrink . Their research offers promise for personalized RNA combination therapies to improve therapeutic response.

Delivering combination therapies

Using the “KP” mouse model, in which a mutant form of the oncogene KRAS is activated and tumor-suppressor gene p53 is deleted, researchers injected mice with RNA-carrying nanoparticles. This mouse model reflects many of the hallmarks of human lung cancer and is often used in preclinical trials. It was originally developed in the laboratory of Koch Institute Director Tyler Jacks, the David H. Koch Professor of Biology, who is co-senior author of this paper.

The nanoparticles are made of a small polymer lipid conjugate; unlike liver-targeting nanoparticles, these preferentially target the lung, and are well-tolerated in the body. They were developed in the laboratories of co-senior author Daniel G. Anderson, the Samuel A. Goldblith Associate Professor of Chemical Engineering, an affiliate of MIT’s Institute of Medical Engineering and Science; and author Robert Langer, the David H. Koch Institute Professor.

In this study, researchers tested the nanoparticle-delivery system with different payloads of therapeutic RNA. They found that delivery of miR-34a, a p53-regulated miRNA, slowed tumor growth, as did delivery of siKRAS, a KRAS-targeting siRNA. Next, researchers treated mice with both miR-34a and siKRAS in the same nanoparticle. Instead of just slowing tumor growth, this combination therapy caused tumors to regress and shrink to about 50 percent of their original size.

Researchers then compared mouse survival time among four treatment options: no treatment; treatment with cisplatin, a small-molecule, standard-care chemotherapy drug; treatment with nanoparticles carrying both miR-34a and siKRAS; and treatment with both cisplatin and the nanoparticles. They found that the nanoparticle treatment extended life just as well as the cisplatin treatment, and furthermore, that the combination therapy of the and cisplatin together extended life by about an additional 25 percent.

Potential for personalized cancer treatments

This early example of RNA combination therapy demonstrates the potential of developing personalized cancer treatments. With efficient delivery of therapeutic RNA, any individual small RNA or combination of RNAs could be deployed to regulate the genetic mutations underlying a given patient’s cancer. Furthermore, these RNA therapies could be combined with more traditional drug therapies for an enhanced effect.

“Small-RNA therapy holds great promise for cancer,” Jacks says. “It is widely appreciated that the major hurdle in this field is efficient delivery to solid tumors outside of the liver, and this work goes a long way in showing that this is achievable.”

“RNA therapies are very flexible and have a lot of potential, because you can design them to treat any type of disease by modifying gene expression very specifically,” says James Dahlman, a graduate student in Anderson’s and Langer’s laboratories who, along with senior postdoc Wen Xue of Jacks’ laboratory, is co-first author of the paper. “We took the best mouse model for lung cancer we could find, we found the best nanoparticle we could use, and for one of the first times, we demonstrate targeted RNA in a clinically relevant model of lung cancer.”

This investigation typifies the Koch Institute’s model of bringing biologists and engineers together to engage in interdisciplinary research.

“This study is a terrific example of the potential of new RNA therapies to treat disease that was done in a highly collaborative way between biologists and engineers,” Langer says. “It’s an example of what makes the Koch Institute very special.”

Contributors to this research from Langer’s and Anderson’s laboratories include postdocs Omar Khan and Gaurav Sahay, former postdoc Avi Schroeder, and Apeksha Dave ’13. Contributors from Jacks’ laboratory include postdoc Tuomas Tammela, Sabina Sood ’13, MIT junior Gillian Yang, and former research technicians Wenxin Cai and Leilani Chirino.

Gene Used In Embryogenesis Can Repair Adult Tissue.


There are some amazing genes and cellular processes active during embryonic development that are never seen again later in life. Though some insects and amphibians are able to carry those traits into adulthood, mammals have a dramatic decrease in the ability to regenerate tissue after birth. A new study has shown that one embryonic protein can be used to help regenerate adult tissue in a living organism, not just in a dish.

The protein Lin28a typically only contributes to processes during embryogenesis, affecting things like metabolism and the pluripotency of stem cells. A study published Nov. 7 in Cell has shown that these proteins can actually be used in adult tissue and help in the regeneration of cartilage, hair follicles, bone, and mesenchyme, a type of undifferentiated connective tissue. It works by binding microRNA in the cell’s nucleus to inhibit let7. Let7 encourages cells to mature and lose the regenerative abilities.

Mice that had been genetically altered to produce Lin28a throughout life had outstanding regenerative power. Though regular mice typically stop producing new hair at around 10 weeks, those with a continued presence of Lin28a kept growing fur throughout their lives. Lin28a also boosted regeneration of limbs. During development, Lin28a is commonly found in the limb buds, but is hardly expressed in those regions after birth. For the mice over expressing Lin28a, some digits that were amputated early in life grew back nearly completely. This ability was diminished as the mouse approached adulthood. Because cardiac tissue also wasn’t regenerated by the presence of Lin28a, there could be other unknown proteins that regulate body aging.

Lin28a was also shown to promote prompt healing of damaged ears, increase metabolism, and contribute to cell proliferation and migration, which are necessary for tissue repair. Unfortunately, some of these attributes can also lead to tumorigenesis, which has been the focus of a great deal of recent cancer research.

This discovery is a long way off from having clinical significance as a miracle “fountain of youth” treatment. Because Lin28a binds to RNA, not the surface of the cell, current drug delivery systems would be very ineffective. Also, because the protein affects so many different tissues in the body, it would be incredibly difficult to target only the desired area. In the future, however, this could be used as a treatment for diseases like alopecia and for tissues that have been injured or are degenerating.

Fountain of youth? Scientists discover why wounds heal quicker for young people


Fountain of youth? Scientists discover why wounds heal quicker for young people .

The mystery of why wounds heal more quickly in the young compared to the elderly may soon be solved following the discovery of two of the genes involved in tissue regeneration.

Scientists believe that the findings will help to develop new drugs and treatments for faster wound-healing as well as shedding light on the ageing process itself, and what could amount to a genetic “fountain of youth”.

Two teams of researchers found separate genes that accelerate tissue regeneration in laboratory mice. Both genes, which are also present in the human genome, are more active in young mice compared to older mice.

The scientists believe that the genes, called Lin28a and IMP1, are designed to be especially active during the foetal stages of development and are gradually turned off as an animal ages – which could explain why wounds take longer to heal in the elderly and how ageing occurs.

One of the teams, led by George Daley of the Boston Children’s Hospital and Harvard Medical School, activated the Lin28a gene in adult mice and found that shaved fur on their backs grew back much faster than in ordinary adult mice where the gene had not be artificially boosted.

“It sounds like science fiction, but Lin28a could be part of a healing cocktail that gives adults the superior tissue repair seen in juvenile animals,” said Dr Daley, whose study is published in the journal Cell.

Asked what the implications are for human health, Dr Daley said: “My strongest conclusion is that Lin28a, or drug manipulations that mimic the metabolic effects of Lin28a, enhances wound healing and tissue repair, and thus in the future might translate into improved healing of wounds after surgery or trauma in patients.”

The study revealed that the Lin28a gene is responsible for a protein that binds to the key molecules of RNA involved in the metabolism of energy within the mitochondria, the “power packs” of the cells. The result is that when the gene is active, the cells are better and more efficient at repairing themselves – the activated genes also accelerated the repair of injuries.

Tissue regeneration is important in early foetal development and when damaged tissues need to be healed. A gradual loss of tissue regeneration and repair is one of the hallmarks of ageing so anything that could improve it could lead to anti-ageing treatments

“We were surprised that what was previously believed to be a mundane cellular ‘housekeeping’ function would be so important for tissue repair,” said Shyh-Chang Ng of Harvard Medical School, the lead author of the Cell study.

“One of our experiments showed that bypassing Lin28a and directly activating mitochondrial metabolism with a small-molecule compound also had the effect of enhancing wound healing, suggesting that it could be possible to use drugs to promote tissue repair in humans.”

The second gene, IMP1, also produces a protein that binds to the RNA molecules, but this time it promotes the self-renewal of key stem cells during foetal development, and also during tissue repair in later life, said Hao Zhu of the University of Texas in Dallas.

“This finding opens up an exciting possibility that metabolism could be modulated to improve tissue repair, whereby metabolic drugs could be employed to promote regeneration,” Dr Zhu said.

Scientists believe that the findings will help to develop new drugs and treatments for faster wound-healing as well as shedding light on the ageing process itself, and what could amount to a genetic “fountain of youth”.

Two teams of researchers found separate genes that accelerate tissue regeneration in laboratory mice. Both genes, which are also present in the human genome, are more active in young mice compared to older mice.

The scientists believe that the genes, called Lin28a and IMP1, are designed to be especially active during the foetal stages of development and are gradually turned off as an animal ages – which could explain why wounds take longer to heal in the elderly and how ageing occurs.

One of the teams, led by George Daley of the Boston Children’s Hospital and Harvard Medical School, activated the Lin28a gene in adult mice and found that shaved fur on their backs grew back much faster than in ordinary adult mice where the gene had not be artificially boosted.

“It sounds like science fiction, but Lin28a could be part of a healing cocktail that gives adults the superior tissue repair seen in juvenile animals,” said Dr Daley, whose study is published in the journal Cell.

Asked what the implications are for human health, Dr Daley said: “My strongest conclusion is that Lin28a, or drug manipulations that mimic the metabolic effects of Lin28a, enhances wound healing and tissue repair, and thus in the future might translate into improved healing of wounds after surgery or trauma in patients.”

The study revealed that the Lin28a gene is responsible for a protein that binds to the key molecules of RNA involved in the metabolism of energy within the mitochondria, the “power packs” of the cells. The result is that when the gene is active, the cells are better and more efficient at repairing themselves – the activated genes also accelerated the repair of injuries.

Tissue regeneration is important in early foetal development and when damaged tissues need to be healed. A gradual loss of tissue regeneration and repair is one of the hallmarks of ageing so anything that could improve it could lead to anti-ageing treatments

“We were surprised that what was previously believed to be a mundane cellular ‘housekeeping’ function would be so important for tissue repair,” said Shyh-Chang Ng of Harvard Medical School, the lead author of the Cell study.

“One of our experiments showed that bypassing Lin28a and directly activating mitochondrial metabolism with a small-molecule compound also had the effect of enhancing wound healing, suggesting that it could be possible to use drugs to promote tissue repair in humans.”

The second gene, IMP1, also produces a protein that binds to the RNA molecules, but this time it promotes the self-renewal of key stem cells during foetal development, and also during tissue repair in later life, said Hao Zhu of the University of Texas in Dallas.

“This finding opens up an exciting possibility that metabolism could be modulated to improve tissue repair, whereby metabolic drugs could be employed to promote regeneration,” Dr Zhu said.

Scientists believe that the findings will help to develop new drugs and treatments for faster wound-healing as well as shedding light on the ageing process itself, and what could amount to a genetic “fountain of youth”.

Two teams of researchers found separate genes that accelerate tissue regeneration in laboratory mice. Both genes, which are also present in the human genome, are more active in young mice compared to older mice.

The scientists believe that the genes, called Lin28a and IMP1, are designed to be especially active during the foetal stages of development and are gradually turned off as an animal ages – which could explain why wounds take longer to heal in the elderly and how ageing occurs.

One of the teams, led by George Daley of the Boston Children’s Hospital and Harvard Medical School, activated the Lin28a gene in adult mice and found that shaved fur on their backs grew back much faster than in ordinary adult mice where the gene had not be artificially boosted.

“It sounds like science fiction, but Lin28a could be part of a healing cocktail that gives adults the superior tissue repair seen in juvenile animals,” said Dr Daley, whose study is published in the journal Cell.

Asked what the implications are for human health, Dr Daley said: “My strongest conclusion is that Lin28a, or drug manipulations that mimic the metabolic effects of Lin28a, enhances wound healing and tissue repair, and thus in the future might translate into improved healing of wounds after surgery or trauma in patients.”

The study revealed that the Lin28a gene is responsible for a protein that binds to the key molecules of RNA involved in the metabolism of energy within the mitochondria, the “power packs” of the cells. The result is that when the gene is active, the cells are better and more efficient at repairing themselves – the activated genes also accelerated the repair of injuries.

Tissue regeneration is important in early foetal development and when damaged tissues need to be healed. A gradual loss of tissue regeneration and repair is one of the hallmarks of ageing so anything that could improve it could lead to anti-ageing treatments

“We were surprised that what was previously believed to be a mundane cellular ‘housekeeping’ function would be so important for tissue repair,” said Shyh-Chang Ng of Harvard Medical School, the lead author of the Cell study.

“One of our experiments showed that bypassing Lin28a and directly activating mitochondrial metabolism with a small-molecule compound also had the effect of enhancing wound healing, suggesting that it could be possible to use drugs to promote tissue repair in humans.”

The second gene, IMP1, also produces a protein that binds to the RNA molecules, but this time it promotes the self-renewal of key stem cells during foetal development, and also during tissue repair in later life, said Hao Zhu of the University of Texas in Dallas.

“This finding opens up an exciting possibility that metabolism could be modulated to improve tissue repair, whereby metabolic drugs could be employed to promote regeneration,” Dr Zhu said.

Detection of Tuberculosis in HIV-Infected and -Uninfected African Adults Using Whole Blood RNA Expression Signatures: A Case-Control Study.


Abstract

Background

A major impediment to tuberculosis control in Africa is the difficulty in diagnosing active tuberculosis (TB), particularly in the context of HIV infection. We hypothesized that a unique host blood RNA transcriptional signature would distinguish TB from other diseases (OD) in HIV-infected and -uninfected patients, and that this could be the basis of a simple diagnostic test.

Methods and Findings

Adult case-control cohorts were established in South Africa and Malawi of HIV-infected or -uninfected individuals consisting of 584 patients with either TB (confirmed by culture ofMycobacterium tuberculosis [M.TB] from sputum or tissue sample in a patient under investigation for TB), OD (i.e., TB was considered in the differential diagnosis but then excluded), or healthy individuals with latent TB infection (LTBI). Individuals were randomized into training (80%) and test (20%) cohorts. Blood transcriptional profiles were assessed and minimal sets of significantly differentially expressed transcripts distinguishing TB from LTBI and OD were identified in the training cohort. A 27 transcript signature distinguished TB from LTBI and a 44 transcript signature distinguished TB from OD. To evaluate our signatures, we used a novel computational method to calculate a disease risk score (DRS) for each patient. The classification based on this score was first evaluated in the test cohort, and then validated in an independent publically available dataset (GSE19491).

In our test cohort, the DRS classified TB from LTBI (sensitivity 95%, 95% CI [87–100]; specificity 90%, 95% CI [80–97]) and TB from OD (sensitivity 93%, 95% CI [83–100]; specificity 88%, 95% CI [74–97]). In the independent validation cohort, TB patients were distinguished both from LTBI individuals (sensitivity 95%, 95% CI [85–100]; specificity 94%, 95% CI [84–100]) and OD patients (sensitivity 100%, 95% CI [100–100]; specificity 96%, 95% CI [93–100]).

Limitations of our study include the use of only culture confirmed TB patients, and the potential that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to their diagnostic group.

Conclusions

In our study, blood transcriptional signatures distinguished TB from other conditions prevalent in HIV-infected and -uninfected African adults. Our DRS, based on these signatures, could be developed as a test for TB suitable for use in HIV endemic countries. Further evaluation of the performance of the signatures and DRS in prospective populations of patients with symptoms consistent with TB will be needed to define their clinical value under operational conditions.

Discussion

We have identified a host blood transcriptomic signature that distinguishes TB from a wide range of OD prevalent in HIV-infected and -uninfected African patients. We found that patients with TB can be distinguished from LTBI with only 27 transcripts and from OD with 44 transcripts. Our findings appear robust as the results are reproducible in both HIV-infected and -uninfected cohorts, in different geographic locations, and in an independent TB patient dataset. The high sensitivity and specificity of the signatures in distinguishing TB from OD, even in the HIV-infected patients that have differing levels of T cell depletion and a wide spectrum of opportunistic infections as well as HIV-related complications, suggests that the signatures are promising biomarkers of TB. The relatively small number of transcripts in our signatures may increase the potential for using transcriptional profiling as a clinical diagnostic tool from a single peripheral blood sample (i.e., using a multiplex assay [35],[36]).

The major challenge for diagnosis of TB in Africa is how to distinguish this disease from the range of other conditions that show similar symptoms in countries where TB and HIV are co-endemic. Previous TB biomarker studies have focused on distinguishing patients with TB from healthy controls, or from LTBI [21],[22],[24], or have used other disease controls that may not represent the “real world” disease spectra from which TB should be clinically differentiated [19],[25]. Furthermore, these TB biomarker studies have also excluded HIV co-infected patients who are the group that most need new diagnostics. Our study design should ensure that our signatures are applicable in TB/HIV endemic countries as we recruited patients with TB concurrently with patients with a range of conditions that present with similar clinical features to TB, as well as recruiting both HIV-infected and -uninfected individuals.

We have identified separate signatures for distinguishing TB/OD and TB/LTBI, which only overlap in three transcripts. In practice the clinical applications of these signatures might be distinct as the TB/LTBI signature would be of value in contact screening, where the concern is distinguishing active disease from previous exposure in minimally symptomatic individuals. The TB/OD signature would be of most value in evaluating symptomatic patients presenting to medical services with symptoms of TB. We have also explored whether a single signature might be used to distinguish TB from both LTBI and OD. The combined signature showed lower performance to the separate TB/LTBI and TB/OD signatures. Further exploration of the operational performance of a combined signature or separate signatures is needed to establish the best strategy.

Although our signatures and DRS distinguished the majority of patients with TB from those with LTBI or OD, a proportion of patients were not correctly classified. There is increasing recognition that TB and LTBI may represent a dynamically evolving continuum, particularly in HIV-infected patients and thus failure to culture M.TB is not absolute proof that TB is not present. Some false assignment by our current “gold standard” is to be expected as noted by post mortem studies at which undiagnosed TB is confirmed [14],[15]. All patients in the OD group presented with symptoms for which TB was included in the differential diagnosis, and it is possible that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to each diagnostic group. Some improvement in sensitivity and specificity of our DRS may also be achieved by weighting the signal from the most discriminatory transcripts, and this could be explored in subsequent refinements of the method.

A major concern in using transcriptional signatures as a clinical diagnostic tool in resource poor settings is the complexity, as well as cost, of the current methodologies. Our results have shown that transcriptional signatures can be used to distinguish TB from OD in an African setting. We explored the feasibility of a simplified method for disease categorization that may facilitate development of a diagnostic test based on our signatures. Our DRS provides a new approach that enables the use of multi-transcript signatures for individual disease risk assignment without the requirement for complex analysis. Our method could be used to develop a simple test in which the transcripts comprising the diagnostic signature (separated into those that are either up- or down-regulated in TB relative to controls) are each measured using a suitable detection system [35], and the combined signature used to identify each patient’s risk of TB. For example, a simple test using the TB/OD signature probes that show increased transcript expression in TB relative to OD could be located in a single well or tube, and those probes that show reduced transcript expression in TB located in a second well or tube. Binding of RNA from a patient’s blood to these probes could be detected as a combined signal from each tube using one of the aforementioned detection systems. To allow normalization, expression of up- or down-regulated transcripts in an individual patient could be compared with that of housekeeping genes, which do not show variation between healthy and disease states. There are methods for rapid detection of multi-transcript signatures including lateral flow reverse transcription (RT)-PCR based systems, nano-pore technology [37], nano-particle enzyme linked detection [38],[39], and detection using nano-wires and electrical impedance [40]. Some of these may be suitable for direct analysis of multiple transcript signatures in blood and at a relatively low cost.

While this study provides a proof of principle that relatively small numbers of RNA transcripts can be used to discriminate active TB from latent TB infection and OD in Africa, limitations remain that need to be addressed in order to translate these results into a clinical test. One such limitation is that our study has not assessed performance of our DRS in patients treated for TB solely on the basis of clinical suspicion, without any microbiological confirmation. Amongst these “probable/possible” patients with TB, there is no gold standard to evaluate any new biomarker. Exclusion of probable/possible patients with TB may have produced better estimates of sensitivity and specificity than would be achieved in a prospective “all comers” study including the entire cohort of patients in whom TB is included in the differential diagnosis. Thus, further evaluation using a prospective population based study in which the decision whether and when to initiate TB treatment is evaluated against the new biomarker is required. Future studies will also be required to refine the use of these biomarkers in a clinical decision process either as an initial screening tool, or in conjunction with more detailed culture based diagnostics.

From a clinical perspective a simple transcriptome-based test that reliably diagnoses or excludes TB in the majority of patients undergoing investigation for suspected TB, using a single blood sample, would be of great value, allowing scarce hospital resources to be focused on the small proportion of patients where the result was indeterminate. The challenge for the academic research community and for industry is to develop innovative methods to translate multi-transcript signatures into simple, cheap tests for TB suitable for use in African health facilities.

SOURCE: PLOS

A Transcriptional Signature for Active TB: Have We Found the Needle in the Haystack?


Analysis of whole-genome RNA expression in human clinical samples is a relatively novel approach to biomarker development. The pattern of RNA expression (i.e., transcriptional signature) can provide a “biological snapshot” of the immune response to physiological stressors, and specific disease states may produce distinct transcriptional signatures. In this week’s issue of PLOS Medicine, Michael Levin and colleagues report that a blood RNA transcriptional signature can be used to diagnose active tuberculosis (TB) in high HIV/TB prevalence settings. Using blood samples from patients referred for TB evaluation at three sites in Cape Town, South Africa (cases from an outpatient TB clinic; controls from two hospitals) and one district hospital in Northern Malawi, the authors identified a minimal set of 44 transcripts that distinguished patients with TB from patients confirmed to have an alternative diagnosis. They then converted the complex expression data into a simple-to-calculate disease risk score, which was highly sensitive (93%, 95% CI [83–100]) and specific (88%, 95% CI [74–97]) for active TB.

Have We Found the Needle in the Haystack?

This landmark study advances the field in several critical ways. For the first time, a blood transcriptional signature for TB was defined by comparison with patients who have conditions that mimic TB in a high burden setting instead of with healthy controls or patients with sarcoidosis or auto-immune diseases [1][4]. The inclusion of controls for whom TB was in the list of differential diagnoses but ultimately excluded increases confidence that the blood transcriptional signature identified may be clinically relevant. Second, the authors developed the disease risk score, which provides a single measure of the degree to which an individual’s RNA expression is consistent with TB. The disease risk score can be calculated by simply subtracting the summed normalized intensities of down-regulated transcripts from those of up-regulated transcripts. By eliminating the need to use complicated bioinformatics to make predictions from the RNA expression data, the disease risk score could simplify the application of transcriptional signatures in clinical settings. Finally, the authors demonstrate convincingly the high diagnostic accuracy of their blood transcriptional signature. The results were impressive in their test set (20% of enrolled patients), including in HIV-infected and smear-negative sub-populations, and in an entirely independent validation dataset published years earlier [1].

Although the results are promising, key questions remain. First, can the results be reproduced in a truly representative population? State-of-the-art technology and bioinformatics are critical tools for identifying prospective targets, but the rigorous application of fundamental epidemiological principles will be indispensable to advancing these technologies into the clinical arena, where it will be necessary to show their utility in truly representative populations. Levin and colleagues describe an “intention-to-test” recruitment strategy but nonetheless enrolled a highly selected patient population. TB patients generally had advanced disease (over 90% of HIV-uninfected patients [96/106] and over 75% of HIV-infected patients [83/109] were smear-positive) and 28% (207 of 751) of patients were excluded because TB status was uncertain. The control group was recruited entirely from inpatient wards and most patients had non-respiratory diseases. These factors resulted in a spectrum bias towards the extremes of disease manifestations, which is known to inflate estimates of diagnostic accuracy [5]. Second, can a robust threshold be developed for the disease risk score that works in different settings? An inherent limitation of the microarray technology used in this study is that it provides only relative quantification of RNA expression so that intensity values are relevant only within, and not across, experiments [6]. Ultimately, to be clinically useful, a threshold will need to be defined a priori rather than on the basis of experimental data and the selected threshold will have to be consistent across geographic settings. Finally, can a platform be developed to enable measurement of the transcriptional signature in low-income countries? A number of novel technologies for quantitative multi-channel measurement of nucleic acid targets are in development. However, the cost and difficulty of assaying a 44-transcript signature seem prohibitive far into the future. Absent a transformative technology, it is difficult to envision transcriptional profiling having a meaningful impact in parts of the world where novel TB diagnostics are most needed [7].

Triage Testing: A Target for Future Research

As Levin and colleagues suggest, their 44-transcript DRS may be more useful as a triage (i.e., rule-out) test [8] because negative predictive value is high (98%, 95% CI [96–100]), while positive predictive value is sub-optimal (66%, 95% CI [46–87]) when TB prevalence is 20%, as is common in routine settings. The concept of a triage test deserves further attention in the TB diagnostics literature. An ideal triage test rules out disease when negative and triggers further testing when positive (e.g., a mammogram for breast cancer screening). Thus, a triage test requires near-perfect sensitivity (particularly when the consequence of missing disease is high) but only moderate specificity. If rapid and inexpensive, such a test could be used to determine which patients presenting with TB symptoms require confirmatory testing (e.g., automated nucleic acid amplification testing or culture) and for TB screening as is recommended in high-risk populations including people living with HIV and household contacts of active TB cases [9],[10].

The 44-transcript signature identified by the authors shows promise as a triage test and might be further optimized for this purpose during its further development. Indeed, it is likely that any set of host-derived biomarkers, especially if based on generic rather than antigen-stimulated immune responses, will have more difficulty achieving high specificity than high sensitivity. In this regard, the commonly used approach of selecting a diagnostic threshold that maximizes the number of correctly classified outcomes is misguided [11]. This cannot lead to a clinically useful test if neither sensitivity nor specificity is high enough to provide meaningful rule-out or rule-in value. Future work to establish a threshold for the 44-transcript signature as a triage test should focus on maximizing sensitivity, even at the cost of decreased specificity. Furthermore, focusing on developing a triage test at the discovery stage may lead to selection of a different set of transcripts that retains 100% sensitivity while providing even better specificity.

Summary

Levin and colleagues have provided compelling proof of the concept that a blood transcriptional signature can distinguish between TB and clinical mimics in high-incidence settings. The field can now move on to asking more practical questions to determine the feasibility and optimal use for an RNA expression-based biomarker for TB in clinical settings. Finding the right signature—the proverbial “needle in the haystack”—may require additional discovery work involving smaller sets of RNA transcripts and will certainly require validation of candidate signatures in diverse settings. Further discovery and validation studies should adhere to fundamental principles of high quality diagnostic evaluations [12], including enrollment of consecutive patients presenting for TB evaluation in representative health facilities. Even if validated, significant technical hurdles remain to translate these important findings to rapid, inexpensive, and simple assays that can impact patient outcomes in countries where TB is most prevalent. The search continues.

Source:PLOS

‘INDIVIDUALIZED’ THERAPY FOR THE BRAIN TARGETS SPECIFIC GENE MUTATIONS CAUSING DEMENTIA AND ALS.


Stem cell-based approach manipulates brain cells in test tube studies

Johns Hopkins scientists have developed new drugs that — at least in a laboratory dish — appear to halt the brain-destroying impact of a genetic mutation at work in some forms of two incurable diseases, amyotrophic lateral sclerosis (ALS) and dementia.

They made the finding by using neurons they created from stem cells known as induced pluripotent stem cells (iPS cells), which are derived from the skin of people with ALS who have a gene mutation that interferes with the process of making proteins needed for normal neuron function.

“Efforts to treat neurodegenerative diseases have the highest failure rate for all clinical trials,” saysJeffrey D. Rothstein, M.D., Ph.D., a professor of neurology and neuroscience at the Johns Hopkins University School of Medicine and leader of the research described online in the journal Neuron. “But with this iPS technology, we think we can target an exact subset of patients with a specific mutation and succeed. It’s individualized brain therapy, just the sort of thing that has been done in cancer, but not yet in neurology.”

Scientists in 2011 discovered that more than 40 percent of patients with an inherited form of ALS and at least 10 percent of patients with the non-inherited sporadic form have a mutation in the C9ORF72 gene. The mutation also occurs very often in people with frontotemporal dementia, the second-most-common form of dementia after Alzheimer’s disease. The same research appeared to explain why some people develop both ALS and the dementia simultaneously and that, in some families, one sibling might develop ALS while another might develop dementia.

In the C9ORF72 gene of a normal person, there are up to 30 repeats of a series of six DNA letters (GGGGCC); but in people with the genetic glitch, the string can be repeated thousands of times. Rothstein, who is also director of the Johns Hopkins Brain Science Institute and the Robert Packard Center for ALS Research, used his large bank of iPS cell lines from ALS patients to identify several with the C9ORF72 mutation, then experimented with them to figure out the mechanism by which the “repeats” were causing the brain cell death characteristic of ALS.

In a series of experiments, Rothstein says, they discovered that in iPS neurons with the mutation, the process of using the DNA blueprint to make RNA and then produce protein is disrupted. Normally, RNA-binding proteins facilitate the production of RNA. Instead, in the iPS neurons with the C9ORF72 mutation, the RNA made from the repeating GGGGCC strings was bunching up, gumming up the works by acting like flypaper and grabbing hold of the extremely important RNA binding proteins, including one known as ADARB2,  needed for the proper production of many other cellular RNAs. Overall, the C9ORF72 mutation made the cell produce abnormal amounts of many other normal RNAs and made the cells very sensitive to stress.

To counter this effect, the researchers developed a number of chemical compounds targeting the problem. This compound behaved like a coating that matches up to the GGGGCC repeats like velcro, keeping the flypaper-like repeats from attracting the bait, allowing the RNA-binding protein to properly do its job.

Rothstein says Isis Pharmaceuticals helped develop many of the studied compounds and, by working closely with the Johns Hopkins teams, could begin testing it in human ALS patients with the C9ORF72 mutation in the next several years. In collaboration with the National Institutes of Health, plans are already underway to begin to identify a group of patients with the C9ORF72 mutation for future research.

Rita Sattler, Ph.D., an assistant professor of neurology at Johns Hopkins and the co-investigator of the study, says without iPS technology, the team would have had a difficult time studying the C9ORF72 mutation. “Typically, researchers engineer rodents with mutations that mimic the human glitches they are trying to research and then study them,” she says. “But the nature of the multiple repeats made that nearly impossible.” The iPS cells did the job just as well or even better than an animal model, Sattler says, in part because the experiments could be done using human cells.

“An iPS cell line can be used effectively and rapidly to understand disease mechanisms and as a tool for therapy development,” Rothstein adds. “Now we need to see if our findings translate into a valuable treatment for humans.”

The researchers also analyzed brain tissue from people with the C9ORF72 mutation who died of ALS. They saw evidence of this bunching up and found that the many genes that were altered as a consequence of this mutation in the iPS cells were also abnormal in the brain tissue, thereby showing that iPS cells can be a faithful tool to study the human disease and discover effective therapies.

In the future, the scientists will look at cerebral spinal fluid from ALS patients with the C9ORF72 mutation, searching for proteins that were found both in the fluid and the iPS cells. These may pave the way to develop markers that can be studied by clinicians to see if the treatment is working once the drug therapy is moved to clinical trials.

ALS, sometimes known as Lou Gehrig’s disease, named for the Yankee baseball great who died from it, destroys nerve cells in the brain and spinal cord that control voluntary muscle movement. The nerve cells waste away or die, and can no longer send messages to muscles, eventually leading to muscle weakening, twitching and an inability to move the arms, legs and body. Onset is typically around age 50 and death often occurs within three to five years of diagnosis. Some 10 percent of cases are hereditary. There is no cure for ALS and there is only one FDA-approved drug treatment, which has just a small effect in slowing disease progression and increasing survival, Rothstein notes.

RNA-based mechanisms underlying axon guidance.


Axon guidance plays a key role in establishing neuronal circuitry. The motile tips of growing axons, the growth cones, navigate by responding directionally to guidance cues that pattern the embryonic neural pathways via receptor-mediated signaling. Evidence in vitro in the last decade supports the notion that RNA-based mechanisms contribute to cue-directed steering during axon guidance. Different cues trigger translation of distinct subsets of mRNAs and localized translation provides precise spatiotemporal control over the growth cone proteome in response to localized receptor activation. Recent evidence has now demonstrated a role for localized translational control in axon guidance decisions in vivo.