A new fluorescent tool for detecting reactive oxygen species based on a chemical found in mushrooms has been developed by scientists at the University of Bath.

Reactive oxygen species (ROS), such as free radicals and peroxides, are produced in cells under oxidative stress. Whilst present in healthy cells in small amounts, excessive ROS in cells are damaging and can lead to cancer and neurodegenerative diseases such as Alzheimer's Disease.

The scientists at Bath, collaborating with researchers in South Korea, have developed a new probe that biologists studying these diseases can use to see changes in cells under the microscope, helping them to understand the fundamental biological processes involving ROS.

They've created a family of new molecules - dubbed AzuFluor - based on azulene, a bright blue chemical found in the mushroom Lactarius indigo. It fluoresces when it comes into contact with a ROS in a one-way reaction, detecting tiny amounts of these reactive oxygen species.

Whilst most fluorescent probes absorb a single photon, AzuFluor absorbs two photons, meaning that two lower energy photons can be used to produce the same level of fluorescence. Using shorter wavelengths of light in infrared range means that the light can penetrate tissues more deeply without harming the cells. This technology has been shown to work in rat tissue; the researchers hope that in the future it could be used as a probe in the human body.

Dr Simon Lewis, Senior Lecturer in the Centre for Sustainable & Circular Technologies (CSCT) at the University of Bath, said: "AzuFluor is a much smaller molecule and simpler to make than other two-photon fluorophores. Its small size makes it easy to diffuse and transport into cells.

"We aim to make a family of these fluorophores that can be used in a range of cell imaging applications."

Professor Tony James, also from the CSCT at Bath, said: "This research has wide-ranging potential applications in cell biology and the pharmaceutical industry and is a great example of a fantastic international collaboration between chemists at Bath and Professor Hwan Myung Kim and his group at Ajou University in South Korea."

Reference:Murfin, L., Weber, M., Park, S., Kim, W., Lopez-Alled, C., & McMullin, C. et al. (2019). Azulene-Derived Fluorescent Probe for Bioimaging: Detection of Reactive Oxygen and Nitrogen Species by Two-Photon Microscopy. Journal Of The American Chemical Society. doi: 10.1021/jacs.9b09813

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Blue Mushroom Dye Used To Develop New Bioimaging Tool - Technology Networks

The genome of human cells looks a lot like a tangled ball of yarn, with tightly wound clumps from which myriad loose strands escape and loop out. But there is order to this tangleand growing evidence that the genomes 3D architecture influences the activity of its genes. Understanding the rules that control gene activity has been the object of a long collaboration between Gladstone investigatorsDeepak Srivastava,Benoit Bruneau,Katherine Pollard,Bruce Conklin, andNevan Krogan, and theirUC San Francisco(UCSF) partnerBrian Black. Together, they have already found many key regulators of gene activity in the heart.

Now, their collaboration has received a strong shot in the arm from the National Institute of Health with the recent award of a Program Project Grant totaling$13 millionbetween the labs for the next five years.

With this new support, the researchers will carry out a comprehensive probe into gene activity in heart cells and its intersection with the genomes 3D organization during heart formation.

It is truly gratifying to see our long collaboration supported in this way by the National Institute of Health,says Srivastava, president of Gladstone Institutes and project leader on this multi-investigator grant. This funding will allow us to dig deep into processes that are fundamental to heart cell biology, but that will also directly inform our efforts to design therapies for congenital heart disease, heart failure, and other heart diseases.

Heart failure is the most common cause of death in adults, and congenital heart defects the most common form of birth defects. These defects have been traced to mutations in a number of proteins that regulate gene activity in heart cells, including the proteins at the core of the researchers new proposal.

However, the investigation of the 3D organization of the genome is a relatively new area, particularly in the heart, says Srivastava, who is also a pediatric cardiologist and has devoted much of his career to understanding heart formation and congenital heart defects.

The work outlined in this grant is therefore expected to yield novel insight into heart disease and spur the design of new therapies. It will also help the researchers improve their ability to coax human cells into becoming various types of heart cells. This technology could eventually be used to regenerate failing heart tissue.

Gladstone Senior InvestigatorBruce Conklinwill lend his expertise in cardiac stem cell biology and CRISPR gene-editing technology to the project.

The researchers plan is to correlate gene activity and genome organization at the whole-genome scale and during multiple stages of heart formation. This will require enormous technological power. It will also require massive computing power and statistical analysis to store and sift through the large data sets the group will generate.

But the team is well-positioned to take on this challenge.

Our studies are facilitated by extraordinary new technology,says Bruneau, also a cardiovascular development specialist and the director of the Gladstone Institute of Cardiovascular Disease.

The$13 millionproposal will leverage Srivastava, Bruneau, and Blacks deep understanding of heart development and disease, and enlist the state-of-the-art technologies and analytic tools that Pollard and Krogan have developed to collect and analyze information about biological networks on a grand scale.

Our team combines a remarkable array of expertise and technologies, says Srivastava, who is also director of the Roddenberry Stem Cell Center at Gladstone. It would be impossible for any one or two labs in isolation to pursue the complex goals we set out to achieve with this project.

Dynamic Protein Networks

The project focuses on a small set of proteins the team has previously shown to be crucial for the formation of a functional heart. These proteins, known as transcription factors, activate or silence genes by binding to specific DNA sequences in the genes vicinity.

The scientists have shown that cardiac transcription factors can associate with each other and with other proteins. Depending on the associations they form, they turn genes on, off, or somewhere in between, and different types of heart cells may form, says Black.

But for a transcription factor to turn a gene on or off, it needs to access the genes DNA sequence. Thats not as easy as it sounds, as much of the genome is wound up in tight coils that give no foothold to transcription factors.

Bruneaus team studies proteins that modulate the accessibility of DNA sequences along the genome, a process known as chromatin remodeling. These proteins unspool segments of the genome from the tightly wound coils, opening up stretches of DNA that transcription factors can bind.

Like transcription factors, chromatin remodeling proteins associate with each other and with other proteins, forming associations that vary depending on the cell type or the stage of heart formation.

Interestingly, Srivastavas group recently discovered that cardiac transcription factors may have long-range effects on the 3D organization of the genome. The genome is housed in a separate compartment of the cell, a spherical structure called the nucleus. Srivastavas team found that cardiac transcription factors may pull genome loops all the way to proteins lining the edges of the nucleus.

The picture that emerges from these findings is that of a vast network of proteins that coordinate gene activity and genome architecture, and change as the heart forms.

Now the researchers want to know how these networks form, how many proteins they entail, and what genes they affect.

Dynamic Lab Partnerships

To answer these questions, the team will analyze the associations between cardiac transcription factors, chromatin remodeling proteins, and their various partners during heart development. They will pair this analysis with a genome-wide survey of the genes these proteins target and of these genes activity.

Our overarching goal is to understand all the levels of gene regulation in developing hearts, from genes and transcription factors to chromatin remodeling and to genome organization within the nucleus, says Bruneau, who is also a professor of pediatrics at UCSF.

The researchers will use a battery of sophisticated techniques to capture the complexes that proteins form with each other or with DNA sequences and to record which genes are active or inactive in different types of heart cells.

They will leverage various models of heart development, including human induced pluripotent stem cells (hiPS cells) that can give rise to heart tissue in the dish, or cells from the developing heart of mouse embryos. They will also use CRISPR technology and other genetic tools to insert mutations in heart cells and evaluate the impact of these mutations on the protein-genome networks.

Their success will depend on high-throughput data collection and analysis, and powerful statistics to guarantee the validity of the findings. Thats where Krogan and Pollard come in.

Krogans labwill contribute technology his lab developed to determine how proteins interact with one another in the celland how those interactions affect the interaction of proteins with DNA.

Pollards groupwill devise statistical methods to rigorously analyze the protein networks and gene activity profiles the researchers uncover through the lens of genetic causes of heart disease.

The biggest challenge will be to develop novel computational methods, including artificial intelligence tools, says Pollard, who directs the Gladstone Institute for Data Science and Biotechnology. This is the first time that scientists will integrate such diverse kinds of data to understand a disease.

Together, these tools will allow the researchers to reliably identify connections between protein networks and gene activity at all stages of heart formation, in the context of disease or healthy heart formation.

This project crystallizes a more than a decade-long collaboration across our labs, with a laser focus on fundamental concepts of gene regulation, says Bruneau.

We will learn how these concepts apply to the heart and to heart diseases, he adds, but we think they will also be relevant to other organs and sets of diseases.

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$13 Million Grant to Probe the Genome of Heart Cells Cardiology2.0 - Cardiology2.0

Liming Wang,1,* Shigao Huang,2,* Shimei Li,1 Ming Li,1 Jun Shi,1 Wen Bai,1 Qianyun Wang,1 Libo Zheng,3 Yongjun Liu3

1Cell Therapy Center, 986 Hospital of Peoples Liberation Army Air Force, Xian, Shaanxi, Peoples Republic of China; 2Cancer Center, Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, Peoples Republic of China; 3Stem Cell Biology and Regenerative Medicine Institution, Yi-Chuang Institute of Bio-Industry, Beijing, Peoples Republic of China

*These authors contributed equally to this work

Correspondence: Shigao HuangCancer Center, Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Room 3013, Building N-22, Taipa, Macau, Peoples Republic of ChinaEmail huangshigao2010@aliyun.comYongjun LiuStem Cell Biology and Regenerative Medicine Institution, Yi-Chuang Institute of Bio-Industry, No. 35, Jinghai 3 Road Economic-Technological Development Area, Beijing, Peoples Republic of ChinaEmail andyliuliu2001@aliyun.com

Background: The traditional anti-inflammation disease-modifying anti-rheumatic drugs (DMARDs) have limited therapeutic effects in rheumatoid arthritis (RA) patients. We previously reported the safety and efficacy of umbilical cord mesenchymal stem cell (UC-MSC) treatment in RA patients that were observed for up to 8 months after UC-MSC infusion. The aim of this study is to assess the long-term efficacy and safety of UC-MSC along with DMARDs for the treatment of RA.Methods: 64 RA patients aged 1864 years were recruited in the study. During the treatment, patients were treated with 40 mL UC-MSC suspension product (2 107 cells/20 mL) via intravenous injection immediately after the infusion of 100 mL saline. The serological markers tests were used to assess safety and the 28-joint disease activity score (DAS28) and the Health Assessment Questionnaire (HAQ) to assess efficacy.Results: 1 year and 3 years after UC-MSC cells treatment, the blood routine, liver and kidney function and immunoglobulin examination showed no abnormalities, which were all in the normal range. The ESR, CRP, RF of 1 year and 3 years after treatment and anti-CCP of 3 years after treatment were detected to be lower than that of pretreatment, which showed significant change (P < 0.05). Health index (HAQ) and joint function index (DAS28) decreased 1 year and 3 years after treatment than before treatment (P < 0.05).Conclusion: UC-MSC cells plus DMARDs therapy can be a safe, effective and feasible therapeutic option for RA patients.

Keywords: rheumatoid arthritis, umbilical cord mesenchymal stem cell, cell therapy

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Efficacy and Safety of Umbilical Cord Mesenchymal Stem Cell Therapy fo | DDDT - Dove Medical Press

Medicines such as insulin for diabetes and clotting factors for hemophilia are hard to synthesize in the lab. Such drugs are based on therapeutic proteins, so scientists have engineered bacteria into tiny protein-making factories. But even with the help of bacteria or other cells, the process of producing proteins for medical or commercial applications is laborious and costly.

Now, researchers at Washington University School of Medicine in St. Louis have discovered a way to supercharge protein production up to a thousandfold. The findings, published Dec. 18 in Nature Communications, could help increase production and drive down costs of making certain protein-based drugs, vaccines and diagnostics, as well as proteins used in the food, agriculture, biomaterials, bioenergy and chemical industries.

The process of producing proteins for medical or commercial applications can be complex, expensive and time-consuming. If you can make each bacterium produce 10 times as much protein, you only need one-tenth the volume of bacteria to get the job done, which would cut costs tremendously. This technique works with all kinds of proteins because it's a basic feature of the universal protein-synthesizing machinery."

Sergej Djuranovic, PhD, associate professor of cell biology and physiology and the study's senior author

Proteins are built from chains of amino acids hundreds of links long. Djuranovic and first author Manasvi Verma, an undergraduate researcher in Djuranovic's lab, stumbled on the importance of the first few amino acids when an experiment for a different study failed to work as expected. The researchers were looking for ways to control the amount of protein produced from a specific gene.

"We changed the sequence of the first few amino acids, and we thought it would have no effect on protein expression, but instead, it increased protein expression by 300%," Djuranovic said. "So then we started digging in to why that happened."

The researchers turned to green fluorescent protein, a tool used in biomedical research to estimate the amount of protein in a sample by measuring the amount of fluorescent light produced. Djuranovic and colleagues randomly changed the sequence of the first few amino acids in green fluorescent protein, generating 9,261 distinct versions, identical except for the very beginning.

The brilliance of the different versions of green fluorescent protein varied a thousandfold from the dimmest to the brightest, the researchers found, indicating a thousandfold difference in the amount of protein produced. With careful analysis and further experiments, Djuranovic, Verma and their collaborators from Washington University and Stanford University identified certain combinations of amino acids at the third, fourth and fifth positions in the protein chain that gave rise to sky-high amounts of protein.

Moreover, the same amino-acid triplets not only ramped up production of green fluorescent protein, which originally comes from jellyfish, but also production of proteins from distantly related species like coral and humans.

The findings could help increase production of proteins not only for medical applications, but in food, agriculture, chemical and other industries.

"There are so many ways we could benefit from ramping up protein production," Djuranovic said. "In the biomedical space, there are many proteins used in drugs, vaccines, diagnostics and biomaterials for medical devices that might become less expensive if we could improve production. And that's not to mention proteins produced for use in the food industry there's one called chymosin that is very important in cheese-making, for example the chemical industry, bioenergy, scientific research and others. Optimizing protein production could have a broad range of commercial benefits."

Source:

Journal reference:

Verma, M., et al. (2019) A short translational ramp determines the efficiency of protein synthesis. Nature Communications. doi.org/10.1038/s41467-019-13810-1.

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Researchers discover new way to supercharge protein production - News-Medical.net

Mount Sinai researchers have found that different mutations in a single gene can have myriad effects on a person's health, suggesting that gene therapies may need to do more than just replenish the missing or dysfunctional protein the gene is supposed to encode, according to a study published in Nature Genetics in November.

"You have to fully understand the mutation to understand how to fix it," said Kristen Brennand, PhD, Associate Professor of Genetics and Genomic Sciences, Neuroscience, and Psychiatry at the Icahn School of Medicine at Mount Sinai, and together with Gang Fang, PhD, Associate Professor of Genetics and Genomic Sciences, one of the lead authors of the study. The two researchers "have been collaborating for seven years on multiple projects that combine our complementary expertise in biology and informatics," said Dr. Fang.

The collaboration originated from Dr. Brennand's interest in the function of the gene neurexin-1, or NRXN1, in psychiatric disorders and Dr. Fang's technology expertise in the use of sophisticated techniques for analyzing different forms of individual genes. Much of the work was led by Shijia Zhu, PhD, formerly a postdoctoral fellow in Dr. Fang's lab, and Erin Flaherty, PhD, a former graduate student in Dr. Brennand's lab.

Patients with schizophrenia, autism, and bipolar disorder sometimes carry mutations in NRXN1. Until now, NRXN1 "had largely been studied only in mice. And, from the mouse studies, we know there are over 300 splice isoforms," said Dr. Brennand. "That means that this one gene makes 300 different proteins in the mouse."

The team set out to understand how NRXN1 functions in typical human neurons, and how different mutations might impact cellular function.

Dr. Brennand and her team started with skin samples from several patients at The Mount Sinai Hospital who had mental health diagnoses and carried mutated forms of the gene. They used these samples, as well as samples from participants without these diagnoses, to culture human induced pluripotent stem cells (hiPSCs)--cells with the ability to grow into any cell in the body.

The cells were then induced to grow into neurons. In the cells that came from patients with mutations in NRXN1, the scientists noted differences in the shape and electrical activity of the neurons as well as the rates at which they matured.

But that wasn't all. All people have two copies of the gene. If there is a mutation, it is usually only in one of those copies. The normal, unmutated gene still produces the healthy protein, but the mutated copy is unable to produce any protein, meaning the individual produces less of the protein than is necessary for normal function. The researchers figured that introducing more of the healthy protein would rescue the neurons, but this wasn't always the case.

Some of the mutations cause the second copy of the gene to produce a separate, mutated version of the protein. The researchers found that these mutated proteins may interfere with the action of the healthy protein. The team found that even cells that could produce enough of the healthy protein that they should have functioned normally would suffer if they were also exposed to a mutant form of the protein--and different mutations led to different problems.

Functionally, these mutant proteins seem to have a dominant negative effect. Overexpression of a single mutant protein in healthy neurons is enough to cause them to fire irregularly."

Dr. Kristen Brennand, Associate Professor, Icahn School of Medicine at Mount Sinai

The study was small, and the gene variants the team studied are rare. In the future it will be important to tease out exactly how the variants impact function: do developmental perturbations lead to later differences in activity or vice versa? But both Dr. Brennand and Dr. Fang emphasized that the overall message is crucial for anyone hoping to use genetics to personalize medicine.

"I went into this really naively, thinking that all patients with deletions in this gene would probably show the same effect," she said. "What we learned is that if you want to move towards precision medicine, it matters not just what genes are impacted, but how they're mutated as well."

Source:

Journal reference:

Flaherty, E., et al. (2019) Neuronal impact of patient-specific aberrant NRXN1 splicing. Nature Genetics. doi.org/10.1038/s41588-019-0539-z.

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Different mutations in a single gene can have myriad effects on a person's health - News-Medical.net