Tag Archives: enzyme

Sharpen Molecular Scissors And Expand The Gene Editing Toolbox

Wake Forest Institute for Regenerative Medicine (WFIRM) scientists have figured out a better way to deliver a DNA editing tool to shorten the presence of the editor proteins in the cells in what they describe as a “hit and run” approach.

CRISPR (clustered regularly interspaced short palindromic repeats) technology is used to alter DNA sequences and modify gene function. CRISPR/Cas9 is an enzyme that is used like a pair of scissors to cut two strands of DNA at a specific location to add, remove or repair bits of DNA. But CRISPR/Cas9 is not 100 percent accurate and could potentially cut unexpected locations, causing unwanted results.

One of the major challenges of CRISPR/Cas9 mRNA technologies is the possibility of off-targets which may cause tumors or mutations,” said Baisong Lu, Ph.D, assistant professor of regenerative medicine at WFIRM and one of the lead authors of the paper. Although other types of lentivirus-like bionanoparticles (LVLPs) have been described for delivering proteins or mRNAs, Lu said, “the LVLP we developed has unique features which will make it a useful tool in the expanding genome editing toolbox.

To address the inaccuracy issue, WFIRM researchers asked the question: Is there a way to efficiently deliver Cas9 activity but achieve transient expression of genome editing proteins? They tested various strategies and then took the best properties of two widely used delivery vehicles – lentivirus vector and nanoparticles – and combined them, creating a system that efficiently packages Cas9 mRNA into LVLPs, enabling transient expression and highly efficient editing.

Lentiviral vector is a widely used gene delivery vehicle in research labs and is already widely used for delivering the CRISPR/Cas9 mRNA technology for efficient genome editing. Nanoparticles are also being used but they are not as efficient in delivery of CRISPR/Cas9.

By combining the transient expression feature of nanoparticle-delivery strategies while retaining the transduction efficiency of lentiviral vectors, we have created a system that may be used for packaging various editor protein mRNA for genome editing in a ‘hit and run’ manner,” said Anthony Atala, M.D., director of WFIRM and co-lead author of the paper. “This system will not only improve safety but also avoid possible immune response to the editor proteins, which could improve in vivo gene editing efficiency which will be useful in research and clinical applications.

The WFIRM team published its findings in a paper published recently in the journal  Nucleic Acids Research.

Source: https://school.wakehealth.edu/

 

New Cheap Test Boosts Detection Of Diseases

Researchers at Queen’s University Belfast have developed a highly innovative new enzyme biomarker test that has the potential to indicate diseases and bacterial contamination saving time, money and possibly lives. The test, developed by scientists at the Institute for Global Food Security at Queen’s, can detect enzyme markers of disease known as proteases in humans, animals and food products.

Proteases are crucial for microorganism growth and are responsible for the progression of many diseasesLevels of proteases can be highly elevated in the urine of patients with diabetic kidney disease, or at the sites of infected wounds. Similarly, in cows, an elevation of proteases in their milk can reveal diseases such as bovine mastitis, a type of mammary gland infection. In food, proteases produced by bacteria contaminated in meat and dairy products can lead to rancidity, as well as decreased shelf life and quality. Current protease detection methods are costly, time-consuming and are not always effective. Scientists at Queen’s Institute for Global Food Security have developed a nanosensor which has resulted in sensitive, fast and cost effective protease detection in milk and urine.

Not only is the test cheap to produce, but it can be used anywhere and is not reliant on laboratory conditions. Eliminating the need to carry out tests in a laboratory setting is life-changing. As well as being cost-effective, it means faster diagnosis,” says Dr Claire McVey, Queen’s researcher and co-author on the study.

The gold-nanoparticle based nanosensor devised by Queen’s researchers indicates when proteases are present through a visible colour-change reactionGold nanoparticles are well known for their capability in speeding up the oxidization of a chemical called tetramethylbenzidine (TMB), visible through a vivid blue-colour formation.

When we add TMB to the casein-covered gold nanoparticles, we can tell virtually instantly if proteases are present by whether or not the solution turns blue. Normally such testing takes much longer,” explains Dr Cuong Cao, the lead academic on the study.

Using this approach, proteases can be detected within 90 minutes without the need for complicated or expensive laboratory equipment.

The findings have been published in the journal Nano Research,

Source: https://www.qub.ac.uk/

New Hope To Fight Alzheimer’s

It is known that the onset of Alzheimer’s disease (AD) is associated with the accumulation of Amyloid beta () peptides in small molecular clusters known as oligomers. These trigger the formation of so-called ‘neurofibrillary tangles’ within neurons hamper their workings, ultimately causing cell death and so significant cognitive decline. Very large Aβ oligomers which form plaques outside neurons, alongside neuroinflammation have also been found to play a key part in the progression of the disease.


The EU-funded iRhom2 in AD project took as its starting point the protein iRhom2, which has been identified as a genetic risk factor for AD due to its pro-inflammatory properties. The team were able to explore further the influence of iRhom2 on neuroinflammation in mice. iRhom2 recently emerged as a protein of note in AD as it aids the maturation of an enzyme called TACE (tumor necrosis factor-α converting enzyme) guiding it towards a cell’s plasma membrane where the enzyme releases a cell-signalling cytokine (TNFα), implicated in the regulation of inflammatory processes. While mice studies have shown that TNFα-dependent inflammation can lead to sepsis and rheumatoid arthritis, it is also thought that the process contributes to neuroinflammatory signalling events, which can cause harm in the brain.

The EU-funded iRhom2 in AD project worked with mice that are prone to develop the hallmarks of AD, amyloid plaques and memory deficits. The team genetically altered iRhom2 in the mice then analysed the progression of the pathology using an array of biochemical and histological methods, together with a number of behavioural tests to assess cognitive decline. The results were somewhat surprising.

We initially hypothesised that iRhom2 would affect one specific aspect of neuroinflammation in AD. What we discovered was even more exciting as it actually affects several different aspects of neuroinflammation simultaneously. So modulating iRhom2 appears particularly well suited to interfere with AD,” explains project coordinator Prof. Dr. Stefan Lichtenthaler.

Source: https://cordis.europa.eu/

Plastic-Eating Enzyme

Scientists have engineered an enzyme which can digest some of our most commonly polluting plastics, providing a potential solution to one of the world’s biggest environmental problems. The discovery could result in a recycling solution for millions of tonnes of plastic bottles, made of polyethylene terephthalate, or PET, which currently persists for hundreds of years in the environment. The research was led by teams at the University of Portsmouth and the US Department of Energy’s National Renewable Energy Laboratory (NREL) and is published in Proceedings of the National Academy of Sciences (PNAS).

 Professor John McGeehan at the University of Portsmouth and Dr Gregg Beckham at NREL solved the crystal structure of PETase—a recently discovered enzyme that digests PET— and used this 3D information to understand how it works. During this study, they inadvertently engineered an enzyme that is even better at degrading the plastic than the one that evolved in nature. The researchers are now working on improving the enzyme further to allow it to be used industrially to break down plastics in a fraction of the time.

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Few could have predicted that since plastics became popular in the 1960s huge plastic waste patches would be found floating in oceans, or washed up on once pristine beaches all over the world. “We can all play a significant part in dealing with the plastic problem, but the scientific community who ultimately created these ‘wonder-materials’, must now use all the technology at their disposal to develop real solutions,” said Professor McGeehan, Director of the Institute of Biological and Biomedical Sciences in the School of Biological Sciences at Portsmouth,

Source: http://uopnews.port.ac.uk/