Single-stranded, noncoding micro-ribonucleic acids (microRNAs), consisting of 18-23 nucleotides, play a key role in regulating gene expression. Levels of microRNAs circulating within blood can be correlated to different states of diseases such as cancer, neurodegenerative disorders and cardiovascular conditions. Many microRNAs within the blood are encapsulated within exosomes, nanoscale vesicles released by the cells.

Accurate measurement of the quantity of microRNAs circulating within the blood is extremely challenging because of their short lengths, similar sequences and low concentration levels. Due to their small number of nucleotides, traditional polymerase chain reaction (PCR) detection methods must necessarily involve a ligation, or linking, step to produce longer complementary DNA strands. Such ligation often produces large biases.

Consequently, large volumes of clinical samples typically required to obtain accurate measurements, but few conventional detection systems can handle this directly without proper sample preparation and volume reduction.
A team of researchers in Italy from the Istituto Italiano di Tecnologia and the University of Naples Federico II, both in Naples, set out to develop a simple, ultrasensitive fluorescence detection system of in-flow microRNAs that uses spectrally encoded microgels.

As the team reports in Biomicrofluidics until now such a multiplexed barcode detection approach has only been performed in time-consuming observation procedures, significantly hindering its possible diagnostic performance.
‘Our technological achievement rests upon the straightforward implementation of a seemingly real-time, microfluidic-based readout of microRNA sequences of interest, handling down to a few microliters of target volume,’ explained Filippo Causa, an associate professor of industrial bioengineering in the Department of Chemical, Materials Engineering, and Industrial Production at the University of Naples Federico II. ‘No previous RNA sequence amplification is required, which reduces evident sources of measurement errors.’
To do this, the researchers first explored a cost-effective and biocompatible non-Newtonian fluid to create the optimal 3-D alignment of microgels in the center of a square-shaped glass capillary.
They then used a simple microfluidic layout to flow the microgel and allow a continuous measurement of the fluorescence signal with several emission wavelengths for the multiplexed barcode detection.

‘We chose microgels with non-overlapping fluorescence-emitting molecules designed to distinguish spectral barcodes for multiplex analysis … and to obtain an absolute quantification of microRNA sequences,’ said Causa. ‘The precise microgel alignment at various throughput rates and an automatic microRNA sequence intensity normalization in flow gives us an opportunity to obtain reliable measurements, similar to quiescent measurement results, without any fundamental pretreatments of the measurement sample.’

To prove their concept of this multiplex spectral microgel analysis within a microfluidic flow, the team used ‘different barcodes corresponding to different emissions at specific wavelengths and the fluorescence intensity of known microRNA concentration,’ which was measured for calibrations of the specific microRNA being explored. Causa said, ‘So far, nine different microgel barcodes have been tested in flow with our detection approach, and more codes are being prepared to multiplex it further.’

As a proof of principle, the team explored microRNA based on its significance to the pathogenesis of various malignant tumors including prostate, gastric, colon, breast and lung cancers.
‘We were able to specifically detect, count and identify in a quasi-real-time manner hundreds of microgels (~80 microgel particles per minute) at sample volumes of only a few microliters,’ said Causa. ‘Our system achieved a microRNA detection limit of 202 femtoMolars in microfluidic flow conditions.’

Measurements were performed with different microgel barcodes and one in particular focused on specific microRNA targets, demonstrating the specificity of the assay for multiplex measurement conditions.
‘A microRNA 21 concentration of 0.74 picoMolars was detected in flow, which is consistent with the initial sample concentration level,’ Causa said. ‘Out of such fluorescence acquisitions, an absolute quantification of the microRNA 21 concentration level was possible.’

In terms of applications for the system, since the specific target detection of microgels can be easily tuned, it can be applied to a wide range of different biomarkers thanks to its barcode structure.
‘Users can also easily adjust its readout speed specifically for any microscopic system,’ said Causa. ‘This means that the system will open up new options for biosensing particles within microfluidic devices.’

PHYS.ORG phys.org/news/2016-12-biomolecules-barcoded-microgels.html

Most prostate cancer (Pca) is diagnosed through a blood test, serum PSA testing. The well appreciated down-side of PSA testing is the diagnosis of a considerable proportion of indolent cancers that are highly unlikely to progress to clinically significant, lethal disease. Our limited ability to accurately identify men destined to suffer and die from the disease from the majority of indolent cases is a major concern and contributes to the dilemma regarding Pca screening and its genetic testing. There is therefore an unmet need to develop genetic tests that can predict whether a man specifically is highly susceptible to the aggressive form of prostate cancer. In some other cancers, such as breast and ovarian cancer, certain predictors of aggressiveness (e.g. BRCA gene mutations) have proven effective in identifying subsets of patients for specific interventions.
In prostate cancer, genetic testing to predict the individual risk to Pca is not performed routinely because of the absence of a marker which accurately identifies aggressive prostate cancer. In a new study, Dr. Alex Zlotta and colleagues identified a new region within the Kallikrein gene, the Kallikrein 6 gene region, detectable in the blood, that is strongly associated with aggressive prostate cancer (defined as Gleason Score ?8) in a cohort of 1858 men from three continents. The team developed a blood test at the Lunenfeld-Tanenbaum Research Institute which detects variants of this Kallikrein 6 gene. The test was validated in three independent cohorts including unique cohorts from large international screening studies for prostate cancer.
The Kallikrein 6 gene variants identified also independently predicted treatment failure after surgery or radiation for prostate cancer in a fourth independent cohort. The frequency of the gene variants varied from 6 to 14% in the population and the increased risk of aggressive prostate cancer was multiplied by almost 3 times in men who harboured the mutations.
Most studies to date have focused on the risk of prostate cancer, not the specific risk of aggressive lethal prostate cancer. The demonstration that germline variants of a new gene, Kallikrein 6, are strongly associated with aggressive prostate cancer, may be of high value in the management of the most common cancer in men.

Lunenfeld-Tanenbaum Research Institute
research.lunenfeld.ca/rssnews/?page=2165

Researchers affiliated with the University of Campinas (UNICAMP) in São Paulo State, Brazil, have shown that genetic information can be used to improve early prediction of the response to drugs in patients with mesial temporal lobe epilepsy (MTLE), one of the most severe forms of epilepsy. Patients who do not respond well to treatment with antiepileptic drugs are candidates for surgery.
The research was conducted at the Brazilian Research Institute for Neuroscience and Neurotechnology (BRAINN) – one of the Research, Innovation and Dissemination Centers (RIDCs) funded by FAPESP – and led by Professor Iscia Lopes-Cendes.
"According to estimates, at the world’s best centres, it takes between 15 and 20 years for patients with MTLE refractory to drug treatment to be referred for surgery," Lopes-Cendes said. "Meanwhile, they continue to suffer from uncontrolled seizures. If we can shorten this process, we can improve the lives of many patients, potentially making the difference between going or not going to university, having or not having a job and a normal life."
MTLE, she explained, is caused by alterations in the functioning of neurons located in the deepest structures of the brain, such as the hippocampus and amygdala, which control important functions such as memory, attention, and anxiety, among others. Seizures due to abnormal electric discharges in a large group of neurons may or may not result in convulsions but do impair memory and other brain functions, often putting the patient at risk of accident and death.
Although MTLE is not the most frequent form of epilepsy, accounting for only 30-40% of cases, it is considered the hardest to treat in adults. Up to 40% of patients with MTLE do not respond to any of the available drugs. In these cases, surgical removal of the brain area that originates the seizures is often recommended.
The aim of the study, according to Lopes-Cendes, was to develop a methodology for distinguishing between the two groups by analysing their genetic material. To do this, the researchers selected a set of 11 genes that have been shown to be involved in antiepileptic drug absorption, metabolism, and transport in the scientific literature.
For these 11 genes, they genotyped 119 different single nucleotide polymorphism (SNP) molecular markers to see which alleles were present.
"We deployed a series of statistical procedures to develop the model with the best capacity to predict whether the patient would be responsive to drug treatment," Lopes-Cendes said. "In this model, we included and excluded variables to see which ones contributed most to the power of the predictors. Besides genetic polymorphisms, we also included clinical data such as the presence or absence of hippocampal atrophy, the age at and frequency of seizures at epilepsy onset, patient gender, and so on."
When only the clinical variables were taken into account, the model’s predictions were about 45% accurate, which, according to Lopes-Cendes, is less than would be achieved by tossing a coin.
However, the model’s accuracy increased to 80% when only SNP markers were used and to 82% when both clinical and genetic variables were used.
As Lopes-Cendes explained, in order to be sure that the two groups of patients belonged to the same population from a genetic standpoint and hence were genuinely comparable, the researchers also genotyped 90 other SNPs in different genes located on the same chromosomes as in the previous analysis.
"We call this testing technique ‘genome control’," she said. "Without it, we risk selecting patient and control groups from different populations, which would invalidate the results of the analysis."
In light of the model’s high level of accuracy, Lopes-Cendes revealed that she and her team at BRAINN now plan to begin a multicentre study involving patients from several countries.
"The idea is to genotype these SNPs at the start of treatment and to follow the patients for two years to see what happens. If the results corroborate our findings in this first study, we’ll have sufficient evidence to include the methodology in clinical practice," she said.

EurekAlert
www.eurekalert.org/pub_releases/2017-04/fda-mie040417.php

24 research studies from the landmark BLUEPRINT project and IHEC consortia reveal how variation in blood cells’ characteristics and numbers can affect a person’s risk of developing complex diseases such as heart disease, and autoimmune diseases including rheumatoid arthritis, asthma, coeliac disease and type 1 diabetes.

The papers, along with another 17 in high-impact journals, represent the culmination of a five-year, £25 million (€30 million) project that brought together 42 leading European universities, research institutes and industry partners and the work of IHEC. The project’s goals were to explore and describe the range of epigenetic changes that take place in bone marrow as stem cells develop into different types of mature blood cell. It also sought to match epigenetic changes and genetic differences to the physical characteristics of each cell type and use this knowledge to understand how these can lead to blood disorders, cancer and other complex diseases*.

In the first study, Sanger Institute researchers worked closely with colleagues at the University of Cambridge and the University of Oxford to carry out the largest and most in-depth study of DNA and blood cell characteristics using the UK BioBank resource and the INTERVAL study. By comparing almost 30 million DNA sequence differences in more than 173,000 people with variation in the physical properties of blood cells the scientists identified 2,500 previously undiscovered locations in the genome that influence blood cell characteristics and functions. Further work showed that genetic differences affecting some of these characteristics are linked to increased risk of heart attack, or to rheumatoid arthritis and other common autoimmune diseases.

“The scale, resolution and homogeneity of our work were vital. Because we examined so many people we were able to discover important ‘rare and low frequency’ genetic differences that are present in fewer than 10 per cent of the population. We found that these can have a much larger impact on the characteristics of blood cells than the common differences studied previously. Of the more than 300 rare and low frequency difference we found, 74 appear to affect the structure of proteins. These give us important clues as to which biological pathways are involved in controlling the production, function and characteristics of blood cells.”

The team found that genetic differences that cause people to have more young red blood cells in their peripheral bloodstreams also increase the risk they will have a heart attack.

“When mature red blood cells rupture in our blood the body replaces them with new, young red cells – a process known as haemolysis. So we think that increased haemolysis and increased risk of coronary heart disease are affected by the same biological pathways. Identifying these pathways may offer new treatment possibilities.”

Dr Adam Butterworth, one of the study’s senior authors, from the University of Cambridge
‘By combining our detailed genetic information with data from the BLUEPRINT project, we were able to identify with high certainty ‘active’ regions of the human genome that are more likely to be involved in disease mechanisms.’

Heather Elding, one of the paper’s first authors, from the Sanger Institute
For example, in another new finding, the research team showed that genetic differences that increased the amount of certain white blood cells, known as eosinophils, also increased the risk of a person developing rheumatoid arthritis, asthma, coeliac disease and type 1 diabetes.

In the second paper, researchers collaborated with scientists at the University of Cambridge, McGill University in Canada and several UK and European institutions to explore the role that epigenetics plays in the development and function of three major human immune cell types: CD14+ monocytes, CD16+ neutrophils and naïve CD4+ T cells, from the genomes of 197 individuals. They studied the contributions of various genetic control mechanisms, including epigenetic changes such as methyl tags on promoter regions in the DNA and histone modifications, to understand how these different levels of regulation interacted with genetic differences to change the expression of genes, immune function and, ultimately, human disease.

The team identified 345 regions of the genome where they could pinpoint the likely molecular causes underlying a person’s predisposition to immune-related diseases such as inflammatory bowel disease, type 1 diabetes and multiple sclerosis.

“We have created an expansive, high-resolution atlas of variations that deepens our understanding of the interplay between the genetic and epigenetic machinery that drives the three primary cells of the human immune system. We have identified hundreds of genetic variations associated with autoimmune diseases that appear to affect the activity of genes in specific regions of the genome, pointing to biological pathways that may be involved in disease and which, ultimately, may be treatable with medication.”

Sanger Institute www.sanger.ac.uk/news/view/landmark-project-shows-heart-disease-and-rheumatoid-arthritis-risk-raised-genetic-changes