Quantum dot technology to advance molecular cell imaging

Researchers from the University of Illinois at Urbana-Champaign bioengineering team and Mayo Clinic have engineered a new type of molecular probe that can measure and count RNA in cells and tissue without organic dyes. The probe is based on the conventional fluorescence in situ hybridization (FISH) technique, but it relies on compact quantum dots to illuminate molecules and diseased cells rather than fluorescent dyes. This research is published in Nature Communications.

Quantum dots illuminate the locations of individual mRNA as red dots in the cytoplasm of a single HeLa cell. The blue region is the nucleus.  Credit: University of Illinois at Urbana-Champaign Department of Bioengineering

Over the last 50 years, fluorescence in situ hybridization technique has evolved into a multi-billion-dollar industry because it effectively images and counts DNA and RNA in single cells. However, fluorescence in situ hybridization technique has its limitations due to the delicate nature of the dyes. For example, the dyes rapidly deteriorate and are not very good at imaging in three dimensions. In addition, conventional fluorescence in situ hybridization technique can only read out a couple of RNA or DNA sequences at a time. Using quantum dots, however, can illuminate the locations of individual mRNA as red dots in the cytoplasm of a single HeLa cell.

The team created unique quantum dots that are made of a zinc, selenium, cadmium, and mercury alloy and are coated with polymers. “The core of the dot dictates the wavelength of emission, and the shell dictates how much light will be given off,” said Smith, who is also affiliated with the Micro + Nanotechnology Lab, Carle Illinois College of Medicine, and Department of Materials Science and Engineering at the University of Illinois.

These dots can emit color independent of the size of the particle, which is not the case for conventional quantum dots. The dots are also small enough (7 nanometers) to fit on a probe that can maneuver between proteins and DNA in a cell, making them more comparable in size to the dyes used in conventional FISH probes.

In experiments with HeLa cells and prostate cancer cells, the researchers found that dye-based FISH cell counts declined rapidly in minutes. The quantum dot-based FISH method provided long-term luminescence to allow counting of RNA for more than 10 minutes, making it possible to acquire 3D cell imaging.

Citation: Liu, Yang, Phuong Le, Sung Jun Lim, Liang Ma, Suresh Sarkar, Zhiyuan Han, Stephen J. Murphy, Farhad Kosari, George Vasmatzis, John C. Cheville, and Andrew M. Smith. “Enhanced mRNA FISH with Compact Quantum Dots.” Nature Communications 9, no. 1 (2018). doi:10.1038/s41467-018-06740-x.

Understanding molecular mechanisms behind germinal matrix hemorrhage

Researchers have utilized a mouse model to determine the molecular mechanisms underlying germinal matrix hemorrhage. Nearly 12,000 premature infants born annually in the US are affected by neonatal brain hemorrhage which results in mortality and long-term morbidity. Unfortunately, no treatment exists for this condition, and the only preventive measure is steroids before birth, which has deleterious effects on brain development. The hemorrhage originates from the rupture of small brain vessels in a highly fragile region known as the germinal matrix.

However, the cellular and molecular mechanisms of this disorder remain poorly understood. In a recent study led by Drs. Jui Dave and Daniel Greif at Yale University found that mutant embryonic mice lacking the gene, Alk5 in pericytes (cells that support small brain vessels) develop germinal matrix hemorrhage. The condition arises due to enhanced proliferation of endothelial cells and upregulated protease activity which result in vessel rupture and hemorrhage. Furthermore, the study reveals that treating mutant mice with a recombinant protein, TIMP3 effectively attenuates the hemorrhage and reduces bleeding. The study is published in the journal Developmental Cell.

The findings from this study provide novel insights into the pathogenesis of germinal matrix hemorrhage and are likely to shed light on other brain disorders such as stroke and aneurysms as well. Although further research is needed to fully understand the beneficial effects of TIMP3, this study promises to broaden the scope of therapeutic intervention for this devastating disorder.

Citation: Dave, Jui M., Teodelinda Mirabella, Scott D. Weatherbee, and Daniel M. Greif. “Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain.” Developmental Cell, 2018. doi:10.1016/j.devcel.2018.01.018.

The role of PRC2 in controlling gene activity in human stem cells.

Researchers at the Babraham Institute have revealed a new understanding of the molecular switches that control gene activity in human embryonic stem cells.

In the developing embryo and during the specialisation of stem cells, the activity of genes must be tightly controlled so that the correct genes are switched on and off at the right time and in the right cells. One of the main ways that this process is regulated is by a protein complex called Polycomb Repressive Complex 2 (PRC2), which keeps genes switched off until they are needed. Earlier research showed that PRC2 is necessary for controlling gene activity during the development of the fruit fly and the mouse. Current research study focusses PRC2’s role in human embryonic stem cells.

As described in the journal Cell Reports, the researchers used the CRISPR gene editing technique to delete PRC2 from human embryonic stem cells. Loss of PRC2 caused the cells to switch on many genes that are not normally active in these cells. These changes led to the inability of embryonic stem cells lacking PRC2 to specialize correctly into mature cell types.

Dr Peter Rugg-Gunn, senior author on the research paper and research group leader at the Babraham Institute explained: “This work is exciting because it reveals that gene activity is controlled by similar molecular switches in human development as in other species such as the fly and mouse. We have also uncovered human-specific differences in the way that embryonic stem cells respond to genes being misregulated. These findings provide new insights into the development of our own species, and might enable new ways to turn embryonic stem cells into useful cell types, such as heart and pancreas, which can be used for cell-replacement therapies.”

References: Collinson, Adam, Amanda J. Collier, Natasha P. Morgan, Arnold R. Sienerth, Tamir Chandra, Simon Andrews, and Peter J. Rugg-Gunn. “Deletion of the Polycomb-Group Protein EZH2 Leads to Compromised Self-Renewal and Differentiation Defects in Human Embryonic Stem Cells.” Cell Reports 17, no. 10 (2016): 2700-714.
doi:10.1016/j.celrep.2016.11.032.
Research funding: The Wellcome Trust, Medical Research Council, Biotechnology and Biological Sciences Research Council.
Adapted from press release by Babraham Institute.

Calorie restriction and cellular aging process

Recent research published in Molecular & Cellular Proteomics offers one glimpse into how cutting calories impacts aging inside a cell. The researchers found that when ribosomes – the cell’s protein makers – slow down, the aging process slows too. The decreased speed lowers production but gives ribosomes extra time to repair themselves. Repairing individual parts of the ribosome on a regular basis enables ribosomes to continue producing high-quality proteins for longer than they would otherwise.

“The ribosome is a very complex machine, sort of like your car, and it periodically needs maintenance to replace the parts that wear out the fastest,” said Brigham Young University biochemistry professor and senior author John Price.

“When tires wear out, you don’t throw the whole car away and buy new ones. It’s cheaper to replace the tires.” So what causes ribosome production to slow down in the first place? At least for mice: reduced calorie consumption.

Price and his fellow researchers observed two groups of mice. One group had unlimited access to food while the other was restricted to consume 35 percent fewer calories, though still receiving all the necessary nutrients for survival. “The calorie-restricted mice are more energetic and suffered fewer diseases,” Price said. “And it’s not just that they’re living longer, but because they’re better at maintaining their bodies, they’re younger for longer as well.”

Despite this study’s observed connection between consuming fewer calories and improved lifespan, Price assured that people shouldn’t start counting calories and expect to stay forever young. Calorie restriction has not been tested in humans as an anti-aging strategy, and the essential message is understanding the importance of taking care of our bodies.

Citation: Mathis, Andrew D., Bradley C. Naylor, Richard H. Carson, Eric Evans, Justin Harwell, Jared Knecht, Eric Hexem, Fredrick F. Peelor, Benjamin F. Miller, Karyn L. Hamilton, Mark K. Transtrum, Benjamin T. Bikman, and John C. Price. “Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals.” Molecular & Cellular Proteomics 16, no. 2 (2016): 243-54. doi:10.1074/mcp.m116.063255
Adapted from press release by Brigham Young University.

Analysis of interactome of Zika virus infected neural cells shows altered expression of more than 500 proteins

Zika virus (ZIKV) interferes with the cellular machinery controlling cell division and alters the expression of hundreds of genes responsible for guiding the formation and development of brain cells, according to findings of research published in Scientific Reports.

Zika virus wikipedia
Zika virus structure. Credit: Wikipedia / David Goodwill

The association between Zika virus (ZIKV) infection and microcephaly has been previously established. Nevertheless, the cellular changes caused by the virus and leading to microcephaly are largely unknown. “Elucidating the foundations of Zika virus infection is crucial in order to develop tools against it”, says Stevens Rehen, the principal investigator of the study and a researcher working at the D’ Or Institute for Research and Education (IDOR) and at the Institute of Biomedical Sciences at Federal University of Rio de Janeiro (UFRJ) in Brazil.

In a previous study published by the group in Science magazine, researchers observed that the pool of human neural stem cells infected by the Brazilian strain of Zika virus was rapidly and completely depleted if compared to non-infected cells. This finding led the group to further investigate how Zika virus disrupts the interactome map (or molecular fingerprinting) of infected cells – which is the entire set of cellular and molecular interactions in a given cell group. The analysis of the interactome of Zika-infected cells may reveal the cellular targets and pathways with which the virus interacts or which it modulates, offering valuable opportunities for drug design.

To this end, human neural cells were infected by a strain of Zika virus (ZIKV) obtained from a Brazilian patient. These cells were then made into neurospheres, which are organized 3D aggregates of neural cells resembling fetal brain tissue that recapitulate many of the normal early and crucial processes that the brain undergoes through development and thus are a great model for studying the human brain. Next, the group identified the molecular fingerprinting of infected and non-infected cells by checking the expression level and status of innumerous genes and proteins.

The analysis revealed that more than 500 proteins in infected neurospheres had their expression level or status (upregulated vs downregulated) altered, if compared to non-infected neurospheres. A number of these altered proteins are normally involved with tasks such as fixing DNA damage or assuring chromosomal stability. Also, proteins that are normally required for cell growth were silent in infected neurospheres, which may explain why Zika-infected cells die much sooner than their non-infected counterparts. Interestingly, genes driving cell specialization were also silent in infected neurospheres, precluding that specialized brain cells were generated. On the other hand, proteins associated with viral replication were over-abundant, most likely the result of a strategy adopted by the virus to promote its own replication in the host cell. A complete list of all human proteins that have been found altered in Zika-infected neurospheres is available in the study entitled “Zika virus disrupts molecular fingerprinting of human neurospheres”, published in Scientific Reports this week. 

According to Patricia Garcez, Assistant Professor at the Federal University of Rio de Janeiro and the first author of the study: “these findings provide insights into the molecular mechanisms of Zika virus (ZIKV) infection over the course of brain development and may explain some of the consequences seen in the brain of newborns with microcephaly”.

Citation: Patricia P. Garcez, Juliana Minardi Nascimento, Janaina Mota de Vasconcelos, Rodrigo Madeiro da Costa, Rodrigo Delvecchio, Pablo Trindade, Erick Correia Loiola, Luiza M. Higa, Juliana S. Cassoli, Gabriela Vitória, Patricia C. Sequeira, Jaroslaw Sochacki, Renato S. Aguiar, Hellen Thais Fuzii, Ana M. Bispo de Filippis, João Lídio da Silva Gonçalves Vianez Júnior, Amilcar Tanuri, Daniel Martins-de-Souza & Stevens K. Rehen. “Zika virus disrupts molecular fingerprinting of human neurospheres.” Scientific Reports 7, Article number: 40780 (2017).
DOI: 10.1038/srep40780
Research funding: Brazilian Development Bank, Funding Authority for Studies and Projects, National Council of Scientific and Technological Development, Foundation for Research Support – State of Rio de Janeiro, São Paulo Research Foundation.
Adapted from press release by D’ Or Institute for Research and Education (IDOR).

Research in mice shows molecular mechanism underlying Oxycodone addiction

RGS9-2, a key signaling protein in the brain known to play a critical role in the development of addiction-related behaviors, acts as a positive modulator of oxycodone reward in both pain-free and chronic pain states, according to a study conducted at the Icahn School of Medicine at Mount Sinai and published in the journal Neuropsychopharmacology. The mechanisms of oxycodone action uncovered through this study will help scientists and physicians develop strategies and tools to dissociate the analgesic (pain relief) actions of opioids from the addiction-related effects.

Pixabay images

Using mouse models of acute and chronic pain, Mount Sinai researchers found that RGS9-2, the intracellular protein that controls the function of opioid receptors in the brain reward center, promotes addiction to oxycodone in pain-free, acute, and chronic pain states. Mice that lacked the gene responsible for encoding RGS9-2 (RGS9KO mice) showed less propensity to develop addiction-related behaviors. Furthermore, the loss of RGS9-2 function does not affect the acute analgesic effects of oxycodone. The research team also found that RSG9-2 plays a protective role towards the development of oxycodone tolerance, as RGS9KO mice became tolerant to the analgesic effects of the drug earlier than those that had the gene. Researchers found that the same mechanisms control sensitivity to oxycodone addiction in pain-free as well as chronic pain states.

Oxycodone is a painkiller that is widely prescribed for acute and chronic pain conditions and is also among the most abused opioids. Oxycodone acts on the same brain receptors as morphine and heroin, the mu opioid receptors, which are present in many areas of the brain that mediate pain relief but are also expressed in the brain network associated with addiction. While there has been an extensive investigation into the mechanisms underlying the analgesia, dependence, and addiction potential of morphine, the mechanism by which oxycodone exerts its actions remained unknown.

“Although oxycodone produces similar analgesic and behavioral effects to those observed with morphine, our study demonstrates that the intracellular actions of morphine and oxycodone are distinct,” says Venetia Zachariou, Ph.D., Associate Professor in the Fishberg Department of Neuroscience and The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai. “Our work reveals that intracellular factors that prevent the actions of morphine may actually promote the actions of oxycodone. This information is particularly important for pain management strategies, as a common course is to have patients oscillate between oxycodone and morphine to achieve pain relief.”

This study provides new information on pathways involved in behavioral responses to oxycodone in pain-free and neuropathic pain states, which will help researchers and clinicians to determine the risks and benefits of oxycodone prescription for the treatment of pain. This knowledge may lead to the development of more efficacious and less addictive compounds for pain management.

Citation: Sevasti Gaspari, Valeria Cogliani, Lefteris Manouras, Ethan M Anderson, Vasiliki Mitsi, Kleopatra Avrampou, Fiona B Carr and Venetia Zachariou. “RGS9-2 Modulates Responses to Oxycodone in Pain-Free and Chronic Pain States.” Neuropsychopharmacology 2017.
DOI: 10.1038/npp.2017.4
Research funding: National Institute of Neurological Disorders and Stroke
Adapted from press release by The Mount Sinai Hospital.