Massachusetts General Hospital (MGH) investigators have used a new sequencing method to identify a group of genes used by the brain’s immune cells – called microglia – to sense pathogenic organisms, toxins or damaged cells that require their response. Identifying these genes should lead to better understanding of the role of microglia both in normal brains and in neurodegenerative disorders and may lead to new ways to protect against the damage caused by conditions like Alzheimer’s and Parkinson’s diseases. The study also finds that the activity of microglia appears to become more protective with ageing, as opposed to increasingly toxic, which some previous studies had suggested.
‘We’ve been able to define, for the first time, a set of genes microglia use to sense their environment, which we are calling the microglial sensome,’ says Joseph El Khoury, MD, of the MGH Center for Immunology and Inflammatory Diseases and Division of Infectious Diseases, senior author of the study. ‘Identifying these genes will allow us to specifically target them in diseases of the central nervous system by developing ways to upregulate or downregulate their expression.’
A type of macrophage, microglia are known to constantly survey their environment in order to sense the presence of infection, inflammation, and injured or dying cells. Depending on the situation they encounter, microglia may react in a protective manner – engulfing pathogenic organisms, toxins or damaged cells – or release toxic substances that directly destroy microbes or infected brain cells. Since this neurotoxic response can also damage healthy cells, keeping it under control is essential, and excess neurotoxicity is known to contribute to the damage caused by several neurodegenerative disorders.
El Khoury’s team set out to define the transcriptome – the complete set of RNA molecules transcribed by a cell – of the microglia of healthy, adult mice and compared that expression profile to those of macrophages from peripheral tissues of the same animals and of whole brain tissue. Using a technique called direct RNA sequencing, which is more accurate than previous methods, they identified a set of genes uniquely expressed in the microglia and measured their expression levels, the first time such a gene expression ‘snapshot’ has been produced for any mammalian brain cell, the authors note.
Since ageing is known to alter gene expression throughout the brain, the researchers then compared the sensome of young adult mice to that of aged mice. They found that – contrary to what previous studies had suggested – the expression of genes involved in potentially neurotoxic actions, such as destroying neurons, was down-regulated as animals aged, while the expression of neuroprotective genes involved in sensing and removing pathogens was increased. El Khoury notes that the earlier studies suggesting increased neurotoxicity with ageing did not look at the cells’ full expression profile and often were done in cultured cells, not in living animals.
‘Establishing the sensome of microglia allows us to clearly understand how they interact with and respond to their environment under normal conditions,’ he explains. ‘The next step is to see what happens under pathologic conditions. We know that microglia become more neurotoxic as Alzheimer’s disease and other neurodegenerative disorders progress, and recent studies have identified two of the microglial sensome genes as contributing to Alzheimer’s risk. Our next steps should be defining the sensome of microglia and other brain cells in humans, identifying how the sensome changes in central nervous system disorders, and eventually finding ways to safely manipulate the sensome pharmacologically.’ Massachusetts General Hospital

Viruses cannot only cause illnesses in humans, they also infect bacteria. Those protect themselves with a kind of ‘immune system’ which – simply put – consists of specific sequences in the genetic material of the bacteria and a suitable enzyme. It detects foreign DNA, which may originate from a virus, cuts it up and thus makes the invaders harmless. Scientists from the Helmholtz Centre for Infection Research (HZI) in Braunschweig have now shown that the dual-RNA guided enzyme Cas9 which is involved in the process has developed independently in various strains of bacteria. This enhances the potential of exploiting the bacterial immune system for genome engineering.
Even though it has only been discovered in recent years the immune system with the cryptic name ‘CRISPR-Cas’ has been attracting attention of geneticists and biotechnologists as it is a promising tool for genetic engineering. CRISPR is short for Clustered Regularly Interspaced Palindromic Repeats, whereas Cas simply stands for the CRISPR-associated protein. Throughout evolution, this molecule has developed independently in numerous strains of bacteria. This is now shown by Prof Emmanuelle Charpentier and her colleagues at the Helmholtz Centre for Infection Research (HZI) who published their finding in the international open access journal Nucleic Acids Research.

The CRISPR-Cas-system is not only valuable for bacteria but also for working in the laboratory. It detects a specific sequence of letters in the genetic code and cuts the DNA at this point. Thus, scientists can either remove or add genes at the interface. By this, for instance, plants can be cultivated which are resistant against vermins or fungi. Existing technologies doing the same thing are often expensive, time consuming or less accurate. In contrast to them the new method is faster, more precise and cheaper, as fewer components are needed and it can target longer gene sequences.

Additionally, this makes the system more flexible, as small changes allow the technology to adapt to different applications. ‘The CRISPR-Cas-system is a very powerful tool for genetic engineering,’ says Emmanuelle Charpentier, who came to the HZI from Umeå and was awarded with the renowned Humboldt Professorship in 2013. ‘We have analysed and compared the enzyme Cas9 and the dual-tracrRNAs-crRNAs that guide this enzyme site-specifically to the DNA in various strains of bacteria.’ Their findings allow them to classify the Cas9 proteins originating from different bacteria into groups. Within those the CRISPR-Cas systems are exchangeable which is not possible between different groups.

This allows for new ways of using the technology in the laboratory: The enzymes can be combined and thereby a variety of changes in the target-DNA can be made at once. Thus, a new therapy for genetic disorders caused by different mutations in the DNA of the patient could be on the horizon. Furthermore, the method could be used to fight the AIDS virus HIV which uses a receptor of the human immune cells to infect them. Using CRISPR-Cas, the gene for the receptor could be removed and the patients could become immune to the virus. However, it is still a long way until this aim will be reached.

Still those examples show the huge potential of the CRISPR-Cas technology. ‘Some of my colleagues already compare it to the PCR,’ says Charpentier. This method, developed in the 1980s, allows scientists to ‘copy’ nucleic acids and therefore to manifold small amounts of DNA to such an extent that they can be analysed biochemically. Without this ground-breaking technology a lot of experiments we consider to be routine would have never been possible. Helmholtz Ceentre for Infection Research

A team of researchers from UCLA and Harvard University have demonstrated a technique that, by measuring the physical properties of individual cells in body fluids, can diagnose cancer with a high degree of accuracy.
The technique, which uses a deformability cytometer to analyse individual cells, could reduce the need for more cumbersome diagnostic procedures and the associated costs, while improving accuracy over current methods. The initial clinical study analysed pleural fluid samples from more than 100 patients.
Pleural fluid, a natural lubricant of the lungs as they expand and contract during breathing, is normally present in spaces surrounding the lungs. Medical conditions such as pneumonia, congestive heart failure and cancer can cause an abnormally large buildup of the fluid, which is called a pleural effusion.
When cytopathologists screen for cancer in pleural effusions, they perform a visual analysis of prepared cells extracted from the fluid. Preparing cells for this analysis can involve complicated and time-consuming dyeing or molecular labelling, and the tests often do not definitively determine the presence of tumour cells. As a result, additional costly tests often are required.
The method in the UCLA–Harvard study, developed previously by the UCLA researchers, requires little sample preparation, relying instead on the imaging of cells as they flow through in microscale fluid conduits.
Imagine squeezing two balloons, one filled with water and one filled with honey. The balloons would feel different and would deform differently in your grip. The researchers used this principle on the cellular level by using a fluid grip to ‘squeeze’ individual cells that are 10,000 times smaller than balloons—a technique called ‘deformability cytometry.’ The amount of a cell’s compression can provide insights about the cell’s makeup or structure, such as the elasticity of its membrane or the resistance to flow of the DNA or proteins inside it. Cancer cells have a different architecture and are softer than healthy cells and, as a result, ‘deform’ differently.
Using deformability cytometry, researchers can analyse more than 1,000 cells per second as they are suspended in a flowing fluid, providing significantly more detail on the variations within each patient’s sample than could be detected using previous physical analysis techniques.
The researchers also noted that the more detailed information they obtained improved the sensitivity of the test: Some patient samples that were not identified as cancerous via traditional methods were found to be so through deformability cytometry. These results were verified six months later.

‘Building off of these results, we are starting studies with many more patients to determine if this could be a cost-effective diagnostic tool and provide even more detailed information about cancer origin,’ said Dino Di Carlo, associate professor of bioengineering at the UCLA Henry Samueli School of Engineering and Applied Science and a co-principal investigator on the research. ‘It could help to reduce laboratory workload and accelerate diagnosis, as well as offer doctors a new way to improve clinical decision-making.’ University of California – Los Angeles

In light of new research, Dr André Lacroix suggests genetic screening to find ‘silent carriers’.
Genetic research suggests to Dr. André Lacroix, professor at the University of Montreal, that clinicians’ understanding and treatment of a form of Cushing’s syndrome affecting both adrenal glands will be fundamentally changed, and that moreover, it might be appropriate to begin screening for the genetic mutations that cause this form of the disease. ‘Screening family members of bilateral adrenal Cushing’s syndrome patients with genetic mutations may identify affected silent carriers,’ Lacroix said ‘The development of drugs that interrupt the defective genetic chemical link that causes the syndrome could, if confirmed to be effective in people, provide individualised specific therapies for hypercortisolism, eliminate the current practice of removing both adrenal glands, and possibly prevent disease progression in genetically affected family members.’ Adrenal glands sit above the kidneys are mainly responsible for releasing cortisol, a stress hormone. Hypercortiolism means a high level of the adrenal hormone cortisol, which causes many symptoms including weight gain, high blood pressure, diabetes, osteoporosis, concentration deficit and increased cardiovascular deaths.

Cushing’s syndrome can be caused by corticosteroid use (such as for asthma or arthritis), a tumour on the adrenal glands, or a pituitary gland that releases too much ACTH. The pituitary gland sits under the brain and releases various hormones that regulate our bodies’ mechanisms.

Jérôme Bertherat is a researcher at Cochin Hospital in Paris. In the study he showed that 55% of Cushing’s Syndrome patients with bilaterally very enlarged adrenal glands have mutations in a gene that predisposes to the development of adrenal tumours. This means that bilateral adrenal Cushing’s is much more hereditary than previously thought. The new knowledge will also enable clinicians to undertake genetic screening. Hervé Lefebvre is a researcher at the University Hospital in Rouen, France. His research shows that the adrenal glands from the same type of patients with two large adrenal glands can produce ACTH, which is normally produced by the pituitary gland. Hormone receptors are the chemical link that cause a cell to behave differently when a hormone is present. Several misplaced hormone receptors cause the ACTH to be produced in the enlarged benign adrenal tissue. Knowing this means that researchers might be able to develop drugs that interrupt the receptors for these hormones and possibly even prevent the benign tissue from developing in the first place. Université de Montréal.

Women who are members of families with BRCA2 mutations but who test negative for the family-specific BRCA2 mutations are still at greater risk for developing breast cancer compared with women in the general population.
Women with certain mutations in their BRCA1 or BRCA2 genes are at increased risk for breast cancer. However, if a woman who comes from a BRCA family tests negative for her family-specific BRCA mutation, her risk for breast cancer is considered to be the same as someone in the general population, according to the National Cancer Institute. This study, however, suggests that it may not always be true.
‘We found that women who test negative for family-specific BRCA2 mutations have more than four times the risk for developing breast cancer than the general population,’ said Gareth R. Evans, M.B.B.S., M.D., M.R.C.P., F.R.C.P., honorary professor of medical genetics and cancer epidemiology at the Manchester Academic Health Science Center at the University of Manchester in the United Kingdom. ‘We also found that any increased risk for breast cancer is largely limited to BRCA2 families with strong family history and other genetic factors.
‘It is likely that these women inherit genetic factors other than BRCA-related genes that increase their breast cancer risk,’ he explained. ‘About 77 single nucleotide polymorphisms [SNPs—genetic variations that can help track the inheritance of disease genes within families] are linked to breast cancer risk. Identification of additional SNPs is necessary to understand why some of the BRCA-negative women from BRCA families are at higher risk.’
The authors note that specialists should use caution when stating that a woman’s breast cancer risk is the same as that of the general population following a negative test, because it may not be true for some women who come from BRCA2 families with a strong family history.
Evans and colleagues used data from the M6-Inherited Cancer in England study, which has screened families of individuals with breast and/or ovarian cancer for mutations in BRCA1 and 2 since 1996. Details on affected individuals, and all tested and untested relatives, were entered into a Filemaker Pro-7 database. From 807 BRCA families, the researchers identified 49 women who tested negative for the family-specific BRCA mutation, but subsequently developed breast cancer. The researchers called these women ‘phenocopies.’
Of the 49 phenocopies identified, 22 were among 279 women who tested negative from BRCA1 families, and 27 were among 251 women who tested negative from BRCA2 families. When the researchers stratified the phenocopies based on their age (30-39, 40-49, 50-59, and 69-80), they found that in each age range there were about twice as many cases of breast cancer as would have been expected from the general population.
Next, to conduct risk analyses, Evans and colleagues calculated the ‘observed versus expected ratio’ (O/E), a ratio of observed risk for breast cancer in BRCA-negative women from BRCA families, versus the risk expected for any woman in the general population.
They found the O/E for phenocopies from BRCA1 families was not substantially higher than that of the general population; however, the O/E for phenocopies from BRCA2 families was 4.57, leading them to conclude that the more than fourfold increased risk for breast cancer among BRCA-negative women is largely limited to BRCA-negative women from BRCA2 families.
When the researchers considered the date of predictive testing (the date on which BRCA testing was done for an individual) instead of the date of family ascertainment (the date the first family member of the individual was referred to genetic service), O/E dropped from 4.57 to 2.01, ‘because there is less follow-up in the predictive test group from time of testing and we may be unaware of breast cancers that have occurred in the near past,’ explained Evans. American Association for Cancer Research