Tiny biomolecular chambers called nanopores that can be selectively heated may help doctors diagnose disease more effectively if recent research by a team at the National Institute of Standards and Technology (NIST), Wheaton College, and Virginia Commonwealth University (VCU) proves effective. Though the findings may be years away from application in the clinic, they may one day improve doctors’ ability to search the bloodstream quickly for indicators of disease—a longstanding goal of medical research.
The team has pioneered work on the use of nanopores—tiny chambers that mimic the ion channels in the membranes of cells—for the detection and identification of a wide range of molecules, including DNA. Ion channels are the gateways by which the cell admits and expels materials like proteins, ions and nucleic acids. The typical ion channel is so small that only one molecule can fit inside at a time.
Previously, team members inserted a nanopore into an artificial cell membrane, which they placed between two electrodes. With this set-up, they could drive individual molecules into the nanopore and trap them there for a few milliseconds, enough to explore some of their physical characteristics.
‘A single molecule creates a marked change in current that flows through the pore, which allows us to measure the molecule’s mass and electrical charge with high accuracy,’ says Joseph Reiner, a physicist at VCU who previously worked at NIST. ‘This enables discrimination between different molecules at high resolution. But for real-world medical work, doctors and clinicians will need even more advanced measurement capability.’
A goal of the team’s work is to differentiate among not just several types of molecules, but among the many thousands of different proteins and other biomarkers in our bloodstream. For example, changes in protein levels can indicate the onset of disease, but with so many similar molecules in the mix, it is important not to mistake one for another. So the team expanded their measurement capability by attaching gold nanoparticles to engineered nanopores, ‘which provides another means to discriminate between various molecular species via temperature control,’ Reiner says.
The team attached gold nanoparticles to the nanopore via tethers made from complementary DNA strands. Gold’s ability to absorb light and quickly convert its energy to heat that conducts into the adjacent solution allows the team to alter the temperature of the nanopore with a laser at will, dynamically changing the way individual molecules interact with it.
‘Historically, sudden temperature changes were used to determine the rates of chemical reactions that were previously inaccessible to measurement,’ says NIST biophysicist John Kasianowicz. ‘The ability to rapidly change temperatures in volumes commensurate with the size of single molecules will permit the separation of subtly different species. This will not only aid the detection and identification of biomarkers, it will also help develop a deeper understanding of thermodynamic and kinetic processes in single molecules.’ EurekAlert

Some diseases are caused by single gene mutations. Current techniques for identifying the disease-causing gene in a patient produce hundreds of potential gene candidates, making it difficult for scientists to pinpoint the single causative gene. Now, a team of researchers led by Rockefeller University scientists have created a map of gene ‘shortcuts’ to simplify the hunt for disease-causing genes.

The investigation, spearheaded by Yuval Itan, a postdoctoral fellow in the St. Giles Laboratory of Human Genetics of Infectious Diseases, has led to the creation of what he calls the human gene connectome, the full set of distances, routes (the genes on the way) and degrees of separation between any two human genes. Itan, a computational biologist, says the computer program he developed to generate the connectome uses the same principles that GPS navigation devices use to plan a trip between two locations. The research is reported in the online early edition of the journal Proceedings of the National Academy of Sciences.
‘High throughput genome sequencing technologies generate a plethora of data, which can take months to search through,’ says Itan. ‘We believe the human gene connectome will provide a shortcut in the search for disease-causing mutations in monogenic diseases.’

Itan and his colleagues, including researchers from the Necker Hospital for Sick Children and the Pasteur Institute in Paris and Ben-Gurion University in Israel, designed applications for the use of the human gene connectome. They began with a gene called TLR3, which is important for resistance to herpes simplex encephalitis, a life-threatening infection from the herpes virus that can cause significant brain damage in genetically susceptible children. Researchers in the St. Giles lab, headed by Jean-Laurent Casanova, previously showed that children with HSE have mutations in TLR3 or in genes that are closely functionally related to TLR3. In other words, these genes are located at a short biological distance from TLR3. As a result, novel herpes simplex encephalitis-causing genes are also expected to have a short biological distance from TLR3.

To test how well the human gene connectome could predict a disease-causing gene, the researchers sequenced exomes – all DNA of the genome that is coding for proteins – of two patients recently shown to carry mutations of a separate gene, TBK1.

‘Each patient’s exome contained hundreds of genes with potentially morbid mutations,’ says Itan. ‘The challenge was to detect the single disease-causing gene.’ After sorting the genes by their predicted biological proximity to TLR3, Itan and his colleagues found TBK1 at the top of the list of genes in both patients. The researchers also used the TLR3 connectome – the set of all human genes sorted by their predicted distance from TLR3 – to successfully predict two other genes, EFGR and SRC, as part of the TLR3 pathway before they were experimentally validated, and applied other gene connectomes to detect Ehlers-Danlos syndrome and sensorineural hearing loss disease causing genes.

‘The human gene connectome is, to the best of our knowledge, the only currently available prediction of the specific route and distance between any two human genes of interest, making it ideal to solve the needle in the haystack problem of detecting the single disease causing gene in a large set of potentially fatal genes,’ says Itan. ‘This can now be performed by prioritising any number of genes by their biological distance from genes that are already known to cause the disease. Rockefeller University

In a small, preliminary study of regular migraine sufferers, scientists have found that measuring a fat-derived protein called adiponectin (ADP) before and after migraine treatment can accurately reveal which headache victims felt pain relief.

A report on the study of people experiencing two to 12 migraine headaches per month, led by researchers at Johns Hopkins, has been published.

‘This study takes the first steps in identifying a potential biomarker for migraine that predicts treatment response and, we hope, can one day be used as a target for developing new and better migraine therapies,’ says study leader B. Lee Peterlin, D.O., an associate professor of neurology and director of headache research at the Johns Hopkins University School of Medicine. She cautioned that larger, confirmatory studies are needed for that to happen.

Experts estimate that roughly 36 million Americans, or 12 percent of the population, suffer from debilitating migraine headaches that last four hours or longer. Migraines are defined as headaches with at least two of four special characteristics: unilateral or one-side-of-the-head occurrence; moderately to severely painful; aggravated by routine activity and of a pounding or throbbing nature. Sufferers generally also feel nauseated or are sensitive to light and sound. Women are three times as likely to get migraines as men.

Such complicated diagnostic criteria mean that diagnosis is tricky, a fact driving efforts, Peterlin says, to find better diagnostic tools.

For the study, Peterlin and her colleagues collected blood from 20 women who visited three headache clinics between December 2009 and January 2012 during an acute migraine attack. Blood was taken before treatment with either sumatriptan/naproxen sodium (a drug routinely given to people with migraines) or a placebo. The investigators re-drew blood at 30, 60 and 120 minutes after the study drug was given. Eleven women received the drug and nine got the placebo.

The researchers measured blood levels of ADP, a protein hormone secreted from fat tissue and known to modulate several of the pain pathways implicated in migraine. The hormone is also implicated in sugar metabolism, insulin regulation, immunity and inflammation, as well as obesity, which is a risk factor for migraines.

Peterlin and her colleagues looked at total adiponectin levels and two subtypes or fragments of total ADP in circulation in the blood: low molecular weight (LMW)-adiponectin and high molecular weight (HMW)-adiponectin. LMW is comprised of small fragments of ADP and it is known to have anti-inflammatory properties, while HMW is made up of larger fragments of ADP and is known to have pro-inflammatory properties. Inflammatory pathways in blood vessels in the head are at work in migraine headache.

The researchers found that in all 20 participants when levels of LMW increased, the severity of pain decreased. When the ratio of HMW to LMW molecules increased, the pain severity increased.

‘The blood tests could predict response to treatment,’ Peterlin says.

At onset of pain – even before study drug was given – the researchers could identify who would be a responder to treatment and who would not, as there was a greater ratio of HMW to LMW in those who would be responders as compared to those who were not.

After study treatment changes in adiponectin were also seen. Interestingly, in those patients who reported less pain after receiving study drug to treat the migraine – whether they got the active migraine medication or a placebo – researchers were able to see a decrease in total levels of ADP in the blood.

Peterlin says the findings indicate it may be possible to develop a treatment that would reduce levels of ADP or parts of adiponectin such as HMW or LMW adiponectin. She says should ADP prove to be a biomarker for migraine, it could help physicians identify who has migraine and know who is likely to respond to which type of medication. It also may help doctors make better medication choices and try alternate drugs sooner. John Hopkins Medicine

Better diagnosis and treatment of cancer could hinge on the ability to better understand a single cell at its molecular level. New research offers a more comprehensive way of analysing one cell’s unique behaviour, using an array of colours to show patterns that could indicate why a cell will or won’t become cancerous.

A University of Washington team has developed a new method for colour-coding cells that allows them to illuminate 100 biomarkers, a ten-time increase from the current research standard, to help analyse individual cells from cultures or tissue biopsies.
‘Discovering this process is an unprecedented breakthrough for the field,’ said corresponding author Xiaohu Gao, a UW associate professor of bioengineering. ‘This technology opens up exciting opportunities for single-cell analysis and clinical diagnosis.’

The research builds on current methods that use a smaller array of colours to point out a cell’s biomarkers – characteristics that indicate a special, and potentially abnormal or diseased, cell. Ideally, scientists would be able to test for a large number of biomarkers, then rely on the patterns that emerge from those tests to understand a cell’s properties.

The UW research team has created a cycle process that allows scientists to test for up to 100 biomarkers in a single cell. Before, researchers could only test for 10 at a time.

The analysis uses quantum dots, which are fluorescent balls of semiconductor material. Quantum dots are the smaller version of the material found in many electronics, including smartphones and radios. These quantum dots are between 2 and 6 nanometers in diameter, and they vary on the colour they emit depending on their size.

Cyclical testing hasn’t been done before, though many quantum dot papers have tried to expand the number of biomarkers tested for in a single cell. This method essentially reuses the same tissue sample, testing for biomarkers in groups of 10 in each round.
‘Proteins are the building blocks for cell function and cell behaviour, but their makeup in a cell is highly complex,’ Gao said. ‘You need to look at a number of indicators (biomarkers) to know what’s going on.’

The new process works like this: Gao and his team purchase antibodies that are known to bind with the specific biomarkers they want to test for in a cell. They pair quantum dots with the antibodies in a fluid solution, injecting it onto a tissue sample. Then, they use a microscope to look for the presence of fluorescent colours in the cell. If they see particular quantum dot colours in the tissue sample, they know the corresponding biomarker is present in the cell.

After completing one cycle, Gao and co-author Pavel Zrazhevskiy, a UW postdoctoral associate in bioengineering, inject a low-pH fluid into the cell tissue that neutralises the colour fluorescence, essentially wiping the sample clean for the next round. Remarkably, the tissue sample doesn’t degrade at all even after 10 such cycles, Gao said.
For cancer research and treatment, in particular, it’s important to be able to look at a single cell at high resolution to examine its details. For example, if 99 percent of cancer cells in a person’s body respond to a treatment drug, but 1 percent doesn’t, it’s important to analyse and understand the molecular makeup of that 1 percent that responds differently.

‘When you treat with promising drugs, there are still a few cells that usually don’t respond to treatment,’ said Gao. ‘They look the same, but you don’t have a tool to look at their protein building blocks. This will really help us develop new drugs and treatment approaches.’ University of Washington

The Red Cross Blood Transfusion Service of Upper Austria has become the first laboratory in Europe to receive accreditation from the European Federation for Immunogenetics (EFI) for the use of human leukocyte antigen (HLA) tests based on next-generation sequencing with Roche’s GS Junior System. This new method will allow more precise and much more rapid tissue-typing and donor selection for stem cell transplants than has been possible to date. In addition, the HLA testing method previously only used for research will now also be available as a standard routine diagnostic procedure.
“Worldwide, around 50,000 people a year urgently require a stem cell transplant, and the chances of finding an allogeneic stem cell donor are about 1:500,000,” said Thomas Schinecker, Head of Roche Sequencing Solutions. “This accreditation is an example of how the potential of next-generation sequencing can be successfully translated from research into medicine and made widely available to patients in areas of high medical need.”
Underlining the benefits of the new standard method, Dr Christian Gabriel, Medical Director of the Red Cross Blood Transfusion Service of Upper Austria, said: “Standardized laboratory procedures are needed to promote positive therapeutic outcomes for patients. EFI accreditation is an important step, allowing large numbers of patients to benefit from the latest technologies.”