Scientists at The Scripps Research Institute (TSRI) and the Salk Institute for Biological Studies have discovered how a mutant protein triggers nerve damage in a subtype of Charcot-Marie-Tooth (CMT) diseases, a group of currently untreatable conditions that cause loss of function in a person’s hands and feet.

The new research suggests future therapies may target this haywire protein, restoring nerve function in patients with CMT.

“This is the first major advancement toward a molecular mechanistic understanding of CMT subtype CMT2D,” said TSRI Professor Xiang-Lei Yang, senior author of the new study with Samuel Pfaff, a neuroscience professor at the Salk Institute and a Howard Hughes Medical Institute investigator. “These findings will help us develop future diagnostics and treatments.”

CMT is one of the most common inherited neurological diseases, affecting about one in 2,500 people. Genetic sequencing usually turns up an array of mutations in people with CMT, making it difficult to pin down the gene responsible and develop a treatment.

In the new study, researchers focused on a protein called glycyl-tRNA synthetase (GlyRS), which is altered in people with disease subtype CMT2D.

Previous work by Yang and her colleagues showed that mutant forms of GlyRS open up their molecular structure to reveal binding components inside—a bit like opening Velcro to reveal the sticky components.

Until now, it was not clear how mutant GlyRS harmed patients.

The work in Yang’s lab, spearheaded by TSRI graduate student Weiwei He, revealed that mutant GlyRS can interact with the Nrp1 receptor on cells. Normally, a growth factor, called vascular endothelial growth factor (VEGF), binds to part of the receptor and relays signals to maintain nerve health.

A postdoctoral researcher in the Yang lab, Huihao Zhou, found that opened-up, mutant GlyRS can bind to the same part of the Nrp1 receptor, blocking the signals for nerve maintenance. This causes motor neurons to decline and even die, breaking the connection between the brain and muscles in the limbs. “GlyRS competes with VEGF,” explained Yang.

Researchers at Salk further confirmed this finding by observing the effect of mutant GlyRS in mouse models of CMT. The team, including Salk Staff Scientist Ge Bai, used gene therapy techniques to ramp up VEGF production in mouse models. Higher levels of VEGF out-competed GlyRS, restoring function in the Nrp1 receptor. The mice with CMT regained some muscle strength and showed significant improvements in CMT symptoms.

“This solves a long-running mystery of how a gene mutation damages the neurons that carry information from the spinal cord to our muscles, resulting in a range of sensory and movement problems,” said Pfaff. “It’s an exciting finding, as we were able in experiments to reduce the symptoms of the disease by targeting the activity of these proteins.”

The next step is to develop targeted strategies that could recognize and intercept GlyRS mutants before they block VEGF. Yang is currently working to screen possible antibodies in collaboration with Kim Janda, the Ely R. Callaway Jr. Professor of Chemistry and member of the Skaggs Institute for Chemical Biology at TSRI.

The new study also has broader implications outside the subtype of CMT examined in these experiments. Yang said mutant GlyRS’s abnormal interaction with Nrp1 is a crucial clue for understanding nerve damage. “This could shed light on the mechanisms behind other forms of CMT,” said Yang. The Scripps Research Institute

Researchers from the University of Illinois at Urbana-Champaign have developed a highly sensitive biosensor based on a differential immuno-capture technology that can detect sub-populations of white blood cells. As part of a small, disposable biochip, the microfluidic biosensor can count CD4+/CD8+ T cells quickly and accurately for AIDS diagnosis in the field.  

“There are 34 million people infected with HIV/AIDS worldwide, many in places that lack testing facilities,” explained Rashid Bashir, an Abel Bliss Professor of Engineering and head of the Department of Bioengineering at Illinois.

“An important diagnostic biomarker for HIV/AIDS is the absolute count of the CD4+ and CD8+ T lymphocytes in the whole blood. The current diagnostic tool—a flow cytometer—is expensive, requires large blood volume, and a trained technician to operate,” Bashir said. “We have developed a microfluidic biosensor based on a differential immuno-capture electrical cell counting technology to enumerate specific cells in 20 minutes using 10 microliters of blood.” (There are about 50 microliters in a drop of blood). 

Human blood is composed of 45 percent of cells with 5 million erythrocytes as compared to only 7000 leukocytes in one microliter of blood. Specific leukocytes like CD4 T cells are of the order of 50-1000 cells per microliter. Electrical cell counting can differentiate cells based on size and membrane properties depending on the frequency of the interrogation signal. However, differentiating cells of same morphology is a challenge.
“For example, a CD4+ T lymphocyte can’t be differentiated from CD4- lymphocytes just by electrical interrogation,” stated Umer Hassan, a postdoctoral researcher in the Bashir’s group and first author of the paper.

“In response to this challenge, we had developed a technique to selectively deplete target leukocytes,” Hassan added. “And our biochip takes whole blood as input, eliminating the need of off-chip sample preparation and effectively reducing the assay time as well.”

In addition to the microfluidic “capture chamber,” the new chip incorporates separate ports for lysing reagents and quenching buffers that preserve the leukocytes for counting by the microfabricated electrodes. Specific leukocytes like CD4 T cells get captured as they interact with the antibodies in the capture chamber; a  second counter recounts the remaining leukocytes. The difference in the respective cell counts give the concentration of the cells captured.

In clinical trials, the differential immuno-capture biochip achieved more than 90 per cent correlation with a flow cytometer for both CD4 T cells for CD8 T cell counts using HIV infected blood samples. The biochip can also be adapted to enumerate other specific cell types such as somatic cells or cells from tissue or liquid biopsies.

The novel biosensor has the potential to be an automated portable blood cell counter for point-of-care applications in developed and resource-limited regions worldwide. Bashir’s group is working on miniaturizing the setup to make the technology handheld, as well as designing a cartridge that can be mass-produced. Engineering at Illinois

Ortho Clinical Diagnostics is emerging stronger than ever since becoming an independent company in 2014, when it was purchased by The Carlyle Group, and has made tremendous progress in developing a range of new assays. Company researchers presented data from five assays currently under development at this year’s American Association for Clinical Chemistry (AACC) meeting.

“We are investing in our business to better serve the modern clinical lab with state-of-the-art solutions,” said Ted Farrell, Vice President Business Field Assays. “We continue to enhance the quality of our products and expand our new product development pipeline for our Clinical Laboratory business.”

The assays presented at AACC address a range of important areas for clinical lab testing including HIV detection and cardiac event monitoring. Following is a quick overview:

• Ortho Clinical Diagnostics is developing a fourth generation assay to detect both HIV 1 antigen genotypes and HIV 1 & 2 antibody subgroups for use on its random access VITROS® systems. The assay demonstrated seroconversion sensitivity consistent with a commercially available fourth generation assay and was more sensitive than a third generation assay.
• A rapid, fully automated, high sensitivity assay is under development for the measurement of cardiac troponin I (cTnl) and is designed to be more analytically sensitive than contemporary cTnl and cTnT assays.
• Preliminary performance data showed that OCD’s prototype VITROS^® Insulin Assay has excellent precision, cross-reactivity with pro-insulin and c-peptide as well as good correlation with two methods that are already commercially available.
• The current VITROS^® Cl- Slide is FDA cleared for use with serum and plasma, but not in urine.Testing of urine samples using the current calibration and the proper testing protocol for plasma and serum resulted in impressive performance, reproducibility and linearity.

Ortho Clinical Diagnostics is focused on bringing targeted solutions like these to its clinical laboratory customers aimed at addressing unmet clinical needs and driving improvements to quality care. It continues to press the boundaries of what’s possible in its quest for new and better assays.  

www.orthoclinical.com

Of the hundreds of genes that can be mutated in a single case of melanoma, only a handful may be true “drivers” of cancer. A Weizmann Institute of Science team has now revealed one of the drivers of a particularly deadly subset of melanomas – one that is still seeing a rise in new cases. This gene is a newly identified member of a group of genes called tumour suppressor genes. It is mutated in some 5.4% of melanomas. Furthermore, its expression was found to be lost in over 30% of human melanomas; and this loss, according to the finding, was associated with reduced patient survival. This discovery might open new doors to understanding how this cancer grows and spreads, and it may lead in the future to new directions in treating this disease.

Prof. Yardena Samuels and her team in the Institute’s Molecular Cell Biology Department were specifically searching for tumour suppressor genes in their database, which consists of more than 500 melanoma genomes and exomes – protein-building sequences – making it the largest melanoma dataset to date. As their name suggests, tumour suppressor genes normally inhibit cell growth, including that of cancer cells. However, when mutated, they act like defective brakes on cellular proliferation. Thus studying these genes is crucial in cancer biology. “The identification of targetable alterations in melanoma is an urgent need. An in-depth understanding of the functional effects of mutations in these genes is the first step toward revealing the underlying mechanism of melanoma growth,” says Dr. Nouar Qutob, a postdoctoral fellow in Samuels’ lab who participated in this research.

Indeed, the melanoma genome sequences contained mutations in known tumour suppressor genes, but there was also a new gene that stood out in the team’s search, named RASA2. The researchers’ next step was to conduct a series of functional experiments to understand exactly what this gene does. They cloned both the normal protein and the most recurrent mutated versions to see their effects on melanoma cells. They found that RASA2 regulates a key protein in the cell, called RAS. RAS has been identified as a major oncogene that contributes to the unchecked growth of cells. When they restored the production of the protein in melanoma cells that harboured RASA2 mutations, these cells stopped growing and eventually died.

Patients with dysfunctional RAS pathways tend to have a worse prognosis than those with other types of melanoma, and, until now, scientists have not managed to create drugs that can target this pathway. “As the RAS pathway is highly dysregulated in cancer, the discovery of an alternative mechanism for its activation is likely to stimulate an avalanche of further research in this field, and is highly likely to have direct clinical relevance. We are now going to focus on RASA2, to find out what proteins it communicates with in healthy cells and melanoma, as well as in the cells’ response to targeted therapy,” says Samuels. Weizmann Insititute

Researchers at the University of Southampton have discovered specific markers on DNA that link the season of birth to risk of allergy in later life.
The season a person is born in influences a wide range of things: from risk of allergic disease, to height and lifespan. Yet little is known about how a one-time exposure like the season of birth has such lasting effects.

The Southampton study conducted epigenetic scanning on DNA samples from a group of people born on the Isle of Wight. They found that particular epigenetic marks (specifically, DNA methylation) were associated with season of birth and still present 18 years later. The research team was also able to link these birth season epigenetic marks to allergic disease, for example people born in autumn had an increased risk of eczema compared to those born in spring. The results were validated in a cohort of Dutch children.

John Holloway, Professor of Allergy and Respiratory Genetics at the University and one of the study’s authors, comments: “These are really interesting results. We know that season of birth has an effect on people throughout their lives. For example generally, people born in autumn and winter are at increased risk for allergic diseases such as asthma. However, until now, we did not know how the effects can be so long lasting.

“Epigenetic marks are attached onto DNA, and can influence gene expression (the process by which specific genes are activated to produce a required protein) for years, maybe even into the next generation. Our study has linked specific epigenetic marks with season of birth and risk of allergy. However, while these results have clinical implications in mediating against allergy risk, we are not advising altering pregnancy timing.”

Dr Gabrielle Lockett, of the University of Southampton and first author of the study, adds: “It might sound like a horoscope by the seasons, but now we have scientific evidence for how that horoscope could work. Because season of birth influences so many things, the epigenetic marks discovered in this study could also potentially be the mechanism for other seasonally influenced diseases and traits too, not just allergy.”

The team say that further research is needed to understand what it is about the different seasons of the year that leads to altered disease risk, and whether specific differences in the seasons including temperature, sunlight levels and diets play a part. More study is also needed on the relationship between DNA methylation and allergic disease, and whether other environmental exposures also alter the epigenome, with potential disease implications. University of Southampton