August 1, 2017

Salk Professor Reuben Shaw has received the National Cancer Institute (NCI) Outstanding Investigator Award (OIA), which encourages cancer research with breakthrough potential. Shaw, a member of Salk's Molecular and Cell Biology Laboratory and holder of the William R. Brody Chair, will receive $4.2 million in direct funding over the next seven years to further his work. The award is granted, according to the NCI website, to innovative cancer researchers with outstanding records of productivity to allow them to take greater risks and be more adventurous in their research.

"It was extremely exciting to get this award as it will provide my lab both the resources and the stability for our ongoing efforts," says Shaw, who is also the director of the Salk Cancer Center, which is one of just seven NCI-designated Basic Research Cancer Centers in the country.

Shaw's research focuses on cancer metabolism: how metabolic pathways are altered in cancer and play a role in the origins and progression of the disease. While investigating one of the most commonly mutated genes in lung cancer, Shaw discovered an energy-sensing pathway that shuts down cell growth and reprograms metabolism when nutrients are scarce. This energy-sensing "starvation" pathway suggests an unexpected and direct link between metabolic pathways and cancer.

His lab went on to molecularly decode a number of new components of this cellular starvation pathway, which connects nutrition and exercise to suppression of both cancer and diabetes. From this work, the lab's studies have led to the discovery of new therapies for cancer and metabolic diseases. Recently, Shaw's lab showed that using a small molecule to target one of the pathways that cells use to synthesize fat can starve cancer cells of the building blocks they need to grow. Previously, he published work showing how different cancers are sensitive to different sources of cellular energy and how a common, deadly lung cancer spreads.

"Reuben's pioneering research points to potential new ways to unravel a variety of cancers and target the disease precisely and effectively," says Salk President Elizabeth Blackburn. "We are delighted that his work is being recognized with this award."

Some of the Shaw lab's ongoing efforts have involved identifying unique metabolic features of tumor cells. Shaw wants to better define different genetic subsets of lung cancer by these features and pinpoint ways to treat them based on that knowledge. "We want to identify the Achilles heel of each tumor subset," he says. "We're not going to treat all lung cancers the same way but rather tailor our attacks based on unique properties of each subtype of cancer. This should yield more effective treatments for all forms of cancer. Our work decoding new components of the energy-sensing pathway has also led to new therapeutic targets for many difference forms of cancer, which alter these same pathways through many different mechanisms."

Shaw is the second Salk scientist in the award's three-year history to be named an NCI Outstanding Investigator. The other is Geoffrey Wahl, a professor in the Gene Expression Laboratory.

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Posted in: Medical Research News | Medical Condition News

Tags: Cancer, Cell, Diabetes, Exercise, Gene, Gene Expression, Genes, Genetic, Genetics, Immunology, Laboratory, Lung Cancer, Metabolism, Molecule, Neuroscience, Nutrients, Nutrition, Tumor

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Salk scientist Reuben Shaw wins NCI Outstanding Investigator Award - News-Medical.net

College Notes appear on a space-available basis. To submit an announcement, email to communities@th-record.com. Put College Notes in the subject line. Announcements can also be mailed to College Notes, Times Herald-Record, P.O. Box 2046, Middletown 10940.

Michael S. Rallo, son of Melissa and Jack Rallo of Chester, graduated summa cum laude from Rutgers University in New Brunswick, N.J., with a Bachelor of Science in Cell Biology and Neuroscience and Exercise Science and Sport Studies. He was named to the dean's list all four years. Rallo will be continuing his education in the MD/PhD program at Rutgers Robert Wood Johnson Medical School and Princeton University.

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College Notes - Times Herald-Record

Structure of a cell | Biology | Science |Khan Academy

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With the goal of improving student learning, a select group of faculty members in Oakland Universitys Department of Biological Sciences attended a national conference dedicated to enhancing teaching methods in the STEM fields. Rasul Chaudhry, Shailesh Lal, Luis Villa-Diaz and Randal Westrick took part in this years Summer Institute on Scientific Teaching, which was held June 4-10 at the University of Minnesota.

The event, which was by invitation only, focused on helping university instructors in the STEM fields create an inclusive environment in which students of all backgrounds and learning styles can succeed.

Chaudhry, who has taught at OU for more than 30 years, said the institute allowed STEM professors to share ideas on how to improve student engagement in science, technology, engineering and mathematics, subjects with a reputation for being academically challenging.

We are always looking for ways to spark students interest. Some students struggle early on (in STEM subjects) and are turned off, Chaudhry said. They may develop a mindset that its too difficult or its just not for me. Our challenge as educators is to reach out to all students and help them see that STEM can be fun and interesting.

During the conference, participants engaged in interactive sessions, worked in small groups with a trained facilitator, and presented instructional materials for feedback and review. OUs team presented a lesson on epigenetics, which is the study of biological mechanisms that control gene expression.

Its a topic that must be taught with sensitivity, Villa-Diaz said, noting that epigenetics play a role in disease predisposition being passed from generation to generation. There could be students in the class who have family history of certain diseases, such as cancer.

The four professors were designated Scientific Teaching Fellows for their dedication to undergraduate education.

Participants discussed ways to maximize student engagement, such as implementing multilingual instruction for non-native speakers and closed captioning for students with hearing impairments. The concept of a flipped classroom in which students watch or listen to a lecture before class, and then engage in discussion and learning exercises during class, was also cited as a way to promote active learning.

Lal noted that an inclusive approach is particularly vital for professors who are teaching students of many different skill levels.

At most universities, professors are teaching a wide spectrum of students, Lal explained. So, were trying to keep the more advanced students interested, while also making sure that no one is left behind.

A students cultural background can also be a pathway to engagement, according to Chaudhry.

Most of the contributions to cell biology came from Caucasians, he said. But there are many other scientific contributions that were made by minorities.

At the conclusion of the institute, participants received a certificate designating them a Scientific Teaching Fellow in recognition of their demonstrated commitment to undergraduate education.

OUs team will be organizing workshops to share what theyve learned with colleagues across campus.

The plan is to spread the message so that we can all use these strategies, Westrick said. We want to attract and retain as many students as possible in STEM. These fields are not only financially rewarding, but also rewarding in terms of their potential to improve peoples lives and make the world a better place.

Financial support for the participants was provided by the Office of the Provost. The institute was jointly sponsored by the Howard Hughes Medical Institute, the Helmsley Charitable Trust and the Yale Center for Teaching and Learning.

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Biology faculty take part in national institute on scientific teaching - News at OU

A close-up of cell mutations that cause cancer. Steve Gschmeissner/Science Source

Chemotherapy is brutal a medicinal atomic bomb that destroys large swaths of cells, both cancerous and normal. And as a result, patients are often left physically devastated.

In a new study published in Science Translational Medicine, scientists at UC Irvine say they've come up with a way to use stem cells to help ameliorate those side effects. Think of it as a surgical strike with cancer-seeking missiles.

Professor Weian Zhao and his colleagues from UC Irvine modified stem cells so that they'd be attracted to enzymes released by breast cancer tumors. So, when injected into the body, the stem cells seek out the cells and bond with them.

The enzymes the scientists identified cause tissue to clump up into bundles of collagen and protein to create stiff tumors. The tumors become lumps that a patient can sometimes feel, and they act as a protective home for the cancerous cells.

The stem cells release an enzyme of their own, in turn, activating a type of chemotherapy that's been injected into the patient, which is inert until in comes in contact with the enzyme. The idea being that the chemotherapy only causes toxicity to a localized area, instead of destroying everything in its path.

"We can use a stem cells to specifically localize and produce the drugs only at the tumor site, so that we can spare the healthy tissue," said Zhao. "So, we can make the treatment more effective and less toxic to the patient."

"I think this is pretty unique in a way that it can target specific metastatic tissues with reduced toxicity overall," said Min Yu, assistant professor at the department of Stem Cell Biology and Regenerative Medicine at USC. "So, in that sense, I think it's very novel and very unique approach."

Yu, who was not involved in the research, complimented the UCI team's methods and results, especially how effective the treatment was on the particular cancer cell that they focused on. However, she said, from patient to patient and cancer to cancer, there are a myriad of different cells responsible, making treatment notoriously difficult to generalize. The therapy isn't a sure thing.

Zhao acknowledged that his team has a while to go before it can prove that the treatment is effective in people. So far, it's only been tested in mice. As a result, FDA approval and human trials could be years away.

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UCI researchers use stem cells as cancer-seeking missiles - 89.3 KPCC

Susana Gonzalez

A once-rising star in stem cell biology who recently lost both her job and a sizable grant has had a fourth paper retracted.

The notice issued by Nature for a 2006 letter cites duplicated images, and a lack of raw data to verify the findings. First author Susana Gonzalez who wasdismissed from her position at the National Center for Cardiovascular Research (CNIC)in Spain last February over allegations of misconduct couldnt be reached by the journal.

Heres the full text of the retraction notice:

In this Letter, some PCR input panels contain duplicated bands (Figs 1b and 2a; and Supplementary Figs 4 and 6a). In Fig. 2e, theARFpromoter panel is a duplicate of the RD panel in Supplementary Fig. 8c. The raw data were not available to verify the data. Given these issues, the authors wish to retract the Letter. The authors deeply regret these errors and apologize to the community. Peter Klatt, Sonia Delgado, Esther Conde, Fernando Lopez-Rios, Montserrat Sanchez-Cespedes, Juan Mendez, Francisco Antequera and Manuel Serrano agree with the Retraction of the Letter. Susana Gonzalez could not be reached.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus has been cited 133 times, according to Clarivate Analytics Web of Science. The letter has been discussed on PubPeer.

Following misconduct allegations, Gonzalez was fired from the CNIC (a decision which she appealed), one of her grants (totaling nearly 2 million Euros)was suspended. Earlier this year, she received two retractions inNature Communications, which also citedfigure duplications and missing raw data. Cell Cycle has also retracted a 2012 paper she co-authored.

Jose F. de Celis, head of theCentre for Molecular Biology Severo Ochoa (CBMSO), where Gonzalez was working in March (but on sick leave), told us:

Susana is not longer at CBMSO, she requested a transfer and we though it was the best option.

Hat tip:Khalid El Bairi

Like Retraction Watch? Consider making atax-deductible contribution to support our growth. You can also follow uson Twitter, like uson Facebook, add us to yourRSS reader, sign up on ourhomepagefor an email every time theres a new post, or subscribe to ourdaily digest. Clickhere to review our Comments Policy. For a sneak peek at what were working on,click here.

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Nir Hacohen, an immunologist and geneticist at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, knew that biology had a problem. He wanted to understand the human immune responses role in cancer and other diseases. But to do that, he first had to address a more fundamental issue: The definition of the immune cell types themselves seemed insufficient, incomplete and outdated.

For over a century, distinctions between types of cells relied on how they appeared under a microscope: their shapes, sizes, locations and their uptake of staining dyes. Recent decades, however, witnessed a shift to molecular methods that use fluorescently labeled antibodies to target protein markers on the cells surface. Although this approach allowed researchers to isolate more cell types, it was not enough, according to Hacohen. Until 2009, biologists could analyze cells only in bulk, averaging signals from multitudes of them to get a picture of what was going on in a tissue. When sequencing RNA from individual cells finally became possible, the initial analyses were what Hacohen called biased and shallow because the few markers used to classify the cells were too insensitive to nuances of differences among them. Does this really capture the complexity of the cell? Hacohen said.

In a study published in Science this past April, he and his team showed that, as expected, much of this complexity had been obscured. Analyzing patterns of gene expression in individual human immune system cells, the researchers refined the definitions of the types known as dendritic cells and monocytes and identified a novel type that had been overlooked. Moreover, they discovered that a cell population thought to comprise one subtype was actually a mixture of two, which perform different functions.

Hacohens work represents one component of a much larger project. Last October, an international community of researchers led by Aviv Regev of the Broad Institute and Sarah Teichmann of the Wellcome Trust Sanger Institute launched the Human Cell Atlas to apply this kind of modeling to the entire body. It aims to catalog not just cell types which are predicted to extend far beyond the 200 types most often cited in textbooks but also the hallmarks of cell types under different conditions and in individuals with different genetic and epigenetic variations. That knowledge is important because it would provide a more comprehensive overview of the dynamic complexity of life. Immune cell subtypes might shift in someone who has an infection or an allergy or an autoimmune disease, for example; or they may vary for different population groups.

This is not comparable to the Human Genome Project, Hacohen said. That was a fairly well-prescribed problem. Here the problem is much more difficult and in a sense encompasses a lot of biology.

The Human Cell Atlas is only one of several projects in molecular and cellular biology looking to synthesize enormous quantities of data to gain deeper insights into just how diverse the cells in our bodies really are, and how complex life is. In 2003, researchers at the KTH Royal Institute of Technology, in Sweden, launched the Human Protein Atlas, which aims to catalog comprehensively the expression, location and spatial distribution of proteins within individual cells. Only within the past few years were members of the project able to start classifying, annotating and analyzing the millions of images they had captured of subcellular structures in different cell types. To reach that point, they first had to spend a decade standardizing, optimizing and scaling up their procedures, which involved using targeted antibodies to stain proteins and then looking for those markers inside healthy and cancerous tissue cells with high-resolution microscopy.

In January 2015, the team charted protein expression across more than 30 human tissues. This past May, they published the second part of their undertaking in Science. Turning their attention to the single-cell level, they mapped more than 12,000 proteins to 30 subcellular structures, in turn defining the proteomes the complete sets of expressed proteins of more than a dozen major organelles. The researchers identified which proteins were found where, explored variations in protein expression from cell to cell and analyzed how cells segregate chemical reactions within themselves.

One of the papers most salient findings, according to its principal investigator, Emma Lundberg, was that as many as half of our proteins can be found in multiple compartments of a cell. Everything that proteins do is specific within the context of their environment, Lundberg said. If one protein is present in the nucleus but also in the plasma membrane, it might have different functions in those compartments.

Take HER2, a receptor protein often overexpressed in certain breast cancers. When found in tumor cell membranes, HER2 correlates with a better prognosis than when it is in the cytoplasm or nucleus. There are more and more and more studies of single proteins showing that this is actually a common phenomenon, Lundberg said. But its the scale of it, she added, that is most exciting.

As much as 50 percent of the proteins that her group observed were expressed in more than one part of a cell. If that figure indicates how big multi-functionality could be, Lundberg said, it makes the cell much more complex and the functionality of the proteome greater.

This heterogeneity offers deeper insights into the fundamentals of protein function, but it may also explain why, for instance, certain drugs result in unwanted side effects.

Another group of scientists, who hope to publish their work in the fall, have been mapping the distribution of proteins in the cell types of the testis home to the greatest number of uniquely expressed protein-coding genes. In doing so, they are reclassifying the cell subtypes that occur during spermatogenesis. Many things are happening in these cells before they become mature, said Cecilia Lindskog Bergstrm of Uppsala University in Sweden, who is collaborating on the research. Proteins that are expressed in a certain sub-stage of sperm development will tell more about the function of these proteins.

This dynamic way of defining cell type is what Hacohen sought to establish further in his study of blood cells. In the findings it reported in May, the Human Protein Atlas began to demonstrate why these refinements may be necessary. The team observed that approximately 15 percent of the proteins exhibited single-cell variation: In a tissue that looked superficially uniform, some cells might differ from their neighbors in the amount or spatial distribution of the proteins they expressed, when one would expect them to be the same. The single-cell RNA sequencing approach of the Human Cell Atlas will allow researchers to create cell profiles based on molecules other than proteins.

In the past, we typically looked at a tissue or an organ in the way youd look at a smoothie, said Bart Deplancke, a biological systems engineer at the cole Polytechnique Fdrale de Lausanne in Switzerland. Based on its overall color and taste, one might assume that a smoothie consists of strawberries and bananas. But that way of looking at it may miss key ingredients and makes it seem as if all parts of the smoothie are identical. With modern techniques, Deplancke said, they can do the tissue-analysis equivalent of looking at a smoothie and saying, I see these different pieces of fruit. And they can see how that full diversity of cell types makes a functional organ. Similarly, they can learn how the full spectrum of cells involved in cancers and other diseases relates to prognosis and recovery.

Deplancke is one of three researchers who have begun organizing the Fly Cell Atlas, which seeks to characterize all the cell types in Drosophila fruit flies. The Allen Institute in Seattle is working toward a similar understanding of the mouse brain. Both hope to apply their findings to explain human behavior and disease, just as the Human Cell Atlas does. Ultimately, integrating the vast datasets generated by these different atlases may prove the greatest challenge of all but, the researchers hope, it will also be the most rewarding, combining structural, genomic and epigenetic approaches under the umbrella of a new kind of cartographic exploration.

Read the rest here:
Cell Atlases Reveal Biology's Frontiers - Quanta Magazine

A series of fluorescence microscopy images detail the blinking behavior of the teams nanoparticle buckyswitches. Credit: Nano Letters 17 (6) pp. 38963901

Visualizing biological cells under a microscope was just made clearer, thanks to research conducted by graduate student Yifei Jiang and principal investigator Jason McNeill of Clemson University's department of chemistry.

With the help of Rhonda Powell and Terri Bruce of Clemson's Light Imaging Facility, the team was able to develop a nanoparticle "switch" that fluoresces to sharpen the resolution of microscopic images that depict small cellular structures. As recently published in Nano Letters, this switch improves upon an imaging method that won the 2014 Nobel Prize in Chemistry.

Because cellular structures emit light at wavelengths smaller than 400-700 nanometers on the electromagnetic spectrum, they often appear blurred through a light microscope. This constraint is referred to as the diffraction limit, and it occurs because of the wave properties of light. As light passes around structures within biological cells, it diffracts, or bends, to a point that light microscopes cannot clearly resolve. The 2014 prize-winning imaging method - single molecule localization microscopy - was invented to surpass this limitation.

"Single molecule localization microscopy is based on molecular 'photoswitches' - fluorescent molecules that you can turn on and off, like a light switch, to beat the diffraction limit," McNeill said. "With this imaging method, the sample is imaged one fluorescent molecule at a time and a computer is used to construct an image that is much sharper than what you could get with a regular light microscope."

The catch, however, is that the fluorescence provided by photoswitches is dim at best, with only a slight improvement in image resolution. Single molecule localization microscopy also requires specialized equipment that can be expensive to obtain.

Cue the "buckyswitch" - the Clemson researchers' enhanced version of a photoswitch. This new type of nanoparticle retains the photoswitch's on-off capability, but is 10 times brighter and easier to use. It also allows microscopes to capture images up to the terapixel level. (That's the equivalent of one trillion pixels, or one million megapixels.)

"These nanoparticles are the first photoswitches to achieve precision down to approximately 1 nanometer, which greatly improves the resolution of super-resolution imaging," Jiang said. "Also, our method only requires one excitation light source, where conventional super-resolution techniques require two lasers; thus, we have simplified the microscope setup."

Jiang assembled the buckyswitch out of a fluorescent, semiconducting conjugated polymer complexed with a chemical derivative of buckminsterfullerene: a soccer-ball-shaped form of carbon.

"The hard part of making a fluorescent nanoparticle that you can turn on and off is that there are lots of areas emitting fluorescence at once," McNeill said. "In the case of fluorescent conjugated polymer, there are dozens or hundreds of chain segments. You can try to make a lot of little switches for each segment, but it's hard to get them all to switch off at the same time. You can't get them synchronized."

By adding the derivative of buckminsterfullerene, called PCBM, to the making of buckyswitches, a "master switch" is formed that regulates the atomic charge of the polymer's segments, thus synchronizing fluorescence. PCBM is able to seize electrons from the polymer segment, giving the segment an overall positive charge. This positive charge reduces the fluorescence of nearby segments, which has a domino effect that turns off fluorescence in the entire nanoparticle.

Bruce - whose background traverses the topics of chemical engineering, applied biology, cell biology, and experience in teaching and industry - likens this imaging method to the view of a suspension bridge at night.

"The wires of the bridge are often illuminated, and when you are standing far away from the bridge, the lights look like one continuous 'rope' of light, instead of individual bulbs. However, if you can make the bulbs blink - such that only every other bulb is 'on' at any time - your eyes can discern the individual bulbs from far away," Bruce said. "The basis for super-resolution microscopy lies in the ability to make fluorescent labels 'blink' just like the lights on the bridge. The work that Dr. McNeill's lab is doing is vital for the advancement of this technology because it focuses on making those individual blinks much brighter, so that our current photon detectors can actually see the blinks. If we can see the blinks with a camera or other photon detector, we can map where the blink occurs, and create an image where we can discern two points of light that are within 10-20 nanometers of one another."

Once the buckyswitch was synthesized, Jiang tested it in E. coli, but not before developing a unique growth media for the bacteria. Typically, E. coli is grown in media that is autofluorescent, meaning that it naturally emits light. Without the proper media, the buckyswitch's fluorescence would be obscured by background light, something that Powell underlined.

"A study like the one Yifei conducted required very little background fluorescence, so I researched media components that would be less likely to be autofluorescent and prepared a 'recipe' for a non-conventional, less autofluorescent nutrient media for bacteria culture," said Powell, who studied both biological sciences and microbiology at Clemson before becoming the research lab manager of the Clemson Light Imaging Facility. Powell and Bruce also worked to provide Jiang with the E. coli for the study.

After all of the necessary components were squared away, Jiang attached the nanoparticle buckyswitches to the surface of E. coli. As hoped, the buckyswitches emitted small flashes of light, which allowed the researchers to determine their precise positions. They then pieced together each flash of light to reconstruct the shape of the E. coli, yielding a super-resolution image.

"We hope this breakthrough will eventually be able to help researchers tackle difficult problems in biology, leading to breakthroughs in the understanding and treatment of disease," the Clemson team said.

The team designed the buckyswitches to work with standard fluorescent microscopes and free software that's available online, making the technology inexpensive and accessible for labs worldwide.

Their publication, titled "Improved Superresolution Imaging Using Telegraph Noise inOrganic Semiconductor Nanoparticles," is featured in the June 14 issue of Nano Letters.

Explore further: Background suppression for super-resolution light microscopy

More information: Yifei Jiang et al, Improved Superresolution Imaging Using Telegraph Noise in Organic Semiconductor Nanoparticles, Nano Letters (2017). DOI: 10.1021/acs.nanolett.7b01440

Journal reference: Nano Letters

Provided by: Clemson University

Excerpt from:
Researchers illuminate the field of microscopy with nanoparticle 'buckyswitch' - Phys.Org

CRISPR-Cas9 gene editing is based on a tactic bacteria developed to protect themselves from viruses.

The Cas9 protein gloms onto a targeted piece of DNA (purple) before cutting it. Bacteria developed Cas9 as aweapon to kill viruses by snipping their DNA, but some viruses came up witha defense, called anti-CRISPR, that inactivates Cas9. The guide RNA (orange)directs Cas9 to its target. (Fuguo Jiang image)

Research now shows that the countermeasure viruses came up with inhibitory proteins referred to as anti-CRISPRs can be used to improve CRISPR-Cas9 as a gene-therapy tool, decreasing off-target gene editing that could cause unwanted side effects.

In a study reported online this week in the journal Science Advances, researchers from UC Berkeley and UC San Francisco show that recently discovered anti-CRISPR proteins decrease off-target effects by as much as a factor of four, acting like a kill switch to disable CRISPR-Cas9 after its done its job.

The study demonstrated that one particular anti-CRISPR protein called AcrIIA4 reduced by four-fold the off-target effects of a CRISPR-Cas9 molecule that uses a guide RNA to find, snip and replace the mutated hemoglobin gene responsible for sickle cell disease. It does this without significantly reducing the desired on-target gene-editing.

Unexpected mutations can arise as a result of off-target gene editing, but our paper like many others shows that off-target effects can be modulated and it is not as serious as people might think, said UC Berkeley postdoctoral fellow Jiyung Jenny Shin, from the lab of Jacob Corn at the Innovative Genomics Institute and one of three first authors of the paper.

In her experiments on human cells in culture, Shin found that delivering CRISPR-Cas9 and then, several hours later, the anti-CRISPR protein, was the most effective way to reduce off-target effects. The protein mimics DNA, glomming onto Cas9, the enzyme that actually cuts the double-stranded DNA, and preventing further cutting.

Even after six hours of effective CRISPR, inserting anti-CRISPR decreases off-target effects by more than two-fold compared to on-target effects, Shin said. Therapeutically, you could treat a patient with CRISPR first, and then treat with anti-CRISPR at a later time and decrease off-target effects.

The researcher who discovered AcrIIA4, Joseph Bondy-Denomy of UC San Francisco, foresees these anti-CRISPR proteins becoming a standard part of CRISPR gene therapy, given along with CRISPR-Cas9 to disable gene editing after a fixed period of time to prevent random off-target cutting.

This Cas9 inhibitor could be encoded on the same piece of DNA as Cas9, for example, precisely timed to turn Cas9 off after the gene editing is done, instead of letting Cas9 linger in the cell and risk off-target effects, said Bondy-Denomy, who is also a co-author of the paper.

Anti-CRISPR bindingThe team included researchers in the lab of Jennifer Doudna, one of the inventors of CRISPR-Cas9 gene editing, who determined how the anti-CRISPR protein binds to the CRISPR-Cas9 complex. Using cryo-electron microscopy, they found that anti-CRISPR essentially mimics DNA, tricking CRISPR-Cas9 into binding with it, and then never letting go.

The anti-CRISPR protein (red on right) mimics DNA, binding in the site where the cutting enzyme Cas9 typically grips the target DNA (left) before it cuts. But the anti-CRISPR protein doesnt let go, essentially killing Cas9s gene editing ability. (Fuguo Jiang image?

The CRISPR inhibitor targets a spot on the Cas9 protein that is so essential for Cas9s function that it cannot operate to cut DNA when its bound by the anti-CRISPR.

Last year, Bondy-Denomy reported finding four anti-CRISPR proteins used by attacking viruses to inactivate the version of the Cas9 protein found in the bacterium Listeria monocytogenes. Two of these also inhibited the Cas9 protein most commonly used by researchers, which is adapted from the bacterium Streptococcus pyogenes and is referred to as SpyCas9. Another team found three other anti-CRISPR proteins that work against a different but promising Cas9 protein adapted from the bacterium Neisseria meningitidis.

The current study looked at the effect of one of the proteins from Listeria, AcrIIA4, on SpyCas9 loaded with a guide RNA that homes in on complementary DNA to bind and cut.

Research at UC Berkeley and elsewhere suggests that CRISPR-Cas9 constantly feints with the cells DNA repair system: as the enzyme cuts at its target site, the cell repairs the DNA, and CRISPR-Cas9 cuts again, repeating this vicious cycle until a mutation arises in the DNA that prevents enzyme binding, at which point the CRISPR-Cas9 molecule moves on to find another binding site.

The current work from the Corn and Doudna labs now suggests that adding an anti-CRISPR after Cas9 has successfully edited a target gene would prevent unintended damage to other portions of a genome.

The ability to turn Cas9 gene editing off is just as important as the ability to turn it on, said Corn, scientific director for biomedicine of the IGI and a UC Berkeley assistant adjunct professor of molecular and cell biology. Imagine if you had an electric razor with no off-switch! For eventual therapeutic applications, it is critical to be able to precisely control when and where gene editing is active. The anti-CRISPR proteins offer opportunities to completely turn off Cas9 as well as fine-tune its activity.Jennys data suggests that there is an ideal time window for letting Cas9 do its job and then turning it off after that amount of time has passed, Bondy-Denomy said. We can actually use the anti-CRISPR proteins as tools to figure out what that time window is, that is, for any one cell type with any one guide RNA sequence, how long we want Cas9 to be active in the cell.

Shin and postdoctoral fellows Fuguo Jiang and Jun-Jie Liu are the three first authors of the paper, which was also co-authored by Benjamin Rauch of UCSF and postdoc Nicolas Bray, researcher Seung Hyun Baik and professor Eva Nogales, in addition to Corn and Doudna, of IGI and UC Berkeleys Department of Molecular and Cell Biology. Doudna and Nogales are Howard Hughes Medical Institute investigators.

The work was supported in part by HHMI, the Li Ka Shing Foundation, the Heritage Medical Research Institute and the National Institute on Aging.

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Anti-CRISPR proteins decrease off-target side effects of CRISPR-Cas9 - UC Berkeley

PUBLISHED: 18:49 12 July 2017 | UPDATED: 18:52 12 July 2017

Paul Brackley

Simon Bullock, cell biology group leader at the MRC LMB in Cambridge, in the fly lab preparing for an open day

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Our cells may be tiny but they are a hive of activity.

Simon Bullock, at the MRC Laboratory of Molecular Biology in Cambridge, is studying what is going on within them.

The department I work in, the cell biology division, is trying to understand how cells and tissues are organised, and there is also a strong interest in how those processes go wrong in human disease, he says.

My research team works on how components are transported within cells by tiny machines called motor proteins. These proteins can walk along tracks within the cell, dragging associated cargo as they go, Simon explains.

Its the cellular equivalent of a railway system, sorting different components to the right places at the right times. This transport process operates in all cells but is particularly important in our nerve cells. Thats because these cells, called neurons, stretch over long distances and consequently rely on a very efficient haulage system within them.

The components being transported are many and various but Simons team have dedicated most of their time to exploring the sorting of ribonucleic acid molecules. These RNAs convey the genetic code in our DNA to another type of machine that reads the code and produces specific proteins from it.

Weve really focused on understanding how these RNAs are targeted to different regions of the cell by motor proteins because that dictates where a protein is made and functions, says Simon.

But the transport system we are studying is important for many other processes in cells. For example, different compartments of the cell need to exchange materials, and this is often done by small membrane-bound structures called vesicles. These vesicles are also moved through the cell by motor proteins.

Moving components around cells by motor proteins, which can take up to 100 steps per second along the track, is much more efficient than having them float around the inside of our cells until they reach their destination.

Each cell has a structure which we call the cytoskeleton, says Simon. Its made up of different types of filaments. As well as providing structural support for the cell, the filaments are used as tracks for the motors.

There are different types of tracks in our cellular railway system some are the equivalent of an inter-city route while others are more akin to a local branch line.

Microtubules are one of the types of tracks. They are used for most long-distance transport in animal cells.

The other kind of track is actin, which is very important in cells for a number of processes. In terms of transport, actin is mostly used for short-distance delivery after cargo has left the microtubules, explains Simon.

The microtubules are hollow tubes, and the motor proteins move along the outside of them.

Some of the motors step in a hand-over-hand fashion, moving in a straight path along one part of the tube. Others appear to have a more chaotic walk, which might allow them to move around obstacles in their path, Simon adds.

While this transport system is essential for normal cell functioning, unfortunately for us it can also be used by some very unpleasant hitchhikers.

We know that the motor proteins in our cells are not just important for trafficking our own cellular components, they are also hijacked by viruses like HIV, rabies and herpes. The viruses have evolved a way to stick to the motors, because this helps them get to where they need to be in the cell, for instance to replicate, says Simon.

One potential long-term benefit of research on motor proteins is that we might have a better understanding of how to block the viral proteins binding to them and thus combat infection.

Viruses can evolve very quickly to prevent a drug binding but this is less of an issue when the virus must preserve the target site in order to interact with a motor in the cell.

Work on motors might also shed light on what goes wrong in neurodegenerative diseases.

One of the earliest things that appears to go wrong in neurodegenerative diseases is transport of cargo along microtubules. It has been speculated that stimulating the transport process could alleviate some of the problems associated with neurodegeneration, says Simon.

However, we are really at the early stages of trying to understand the basic biology of how transport processes work, and translating the results into medicines will be challenging and take a long time.

Simons team use a range of techniques to study the basic biology of transport processes. One line of research involves fruit flies, which can have their genes changed very quickly and easily.

We have been doing a lot of imaging of the cells of fruit flies, says Simon. Part of that has involved labelling cargoes to make them fluorescent, which includes using a protein initially identified in jellyfish by other researchers. We fuse the fluorescent protein to the cargo and then we can use our microscopes to watch it being moved by motors with the cell.

Simons colleagues at the MRC Laboratory of Molecular Biology have also made use of the institutes 5million cryo-electron microscopes to help them put together the first complete 3D model of one of these tiny motors, known as dynein.

This family of motor proteins move along microtubules to transport cargoes, including proteins and RNAs, to different parts of our cells.

Dynein is also known to be involved with many diseases, including viral infections.

Andrew Carters group in the LMBs structural studies division collaborated with Alexander Birds group at the Max Planck Institute in Dortmund on the work.

On its own, dynein does not move for long distances it acts like a train with its brakes on. But once bound to a protein complex called dynactin and proteins on the cargo, it forms a formidable transport machine capable of taking thousands of steps without stopping. Disrupting this process can cause defects in the formation of the brain, leading to learning disabilities or certain forms of epilepsy. The work by Andrew and his colleagues shows how dynein is held in an inactive state and how it is triggered to move only after the cargo is loaded.

Research at the taxpayer-funded Medical Research Council facility is driven by a remit to improve our understanding of human biology and the lab, which last month attracted 2,000 people to an open day at its Cambridge Biomedical Campus home, has an incredible 10 Nobel Prizes to its name.

A lot of major scientific discoveries have been driven by curiosity, observes Simon. People often didnt set out to make a specific discovery but they followed their interests. They saw something unexpected and that led to a completely new line of research. Curiosity-driven research continues to be really important.

Nonetheless, the benefits of translating this research into treatments through collaborating with pharmaceutical companies are clear. With AstraZeneca building its global HQ and R&D facility over the road from the LMB, the opportunities for collaboration will only increase. LMB and AstraZeneca have already been collaborating closely on some projects, as the Cambridge Independent has reported.

I think having AstraZeneca as our neighbours will be fantastic, says Simon. The expertise at AstraZeneca and LMB are highly complementary and there is lots of room for synergy.

Bringing microscopic life into our schools to inspire pupils

Simon Bullock was drawn into biology as a teenager by what he observed when he peered down a microscope.

Rather than learning details of how biochemical reactions work, it was seeing a water fleas heartbeat that really got me hooked. And I still love looking down a microscope in my work, he says.

Simon and colleagues at the MRC Laboratory of Molecular Biology have developed a project that aims to offer younger children the opportunity to be amazed by life at a microscopic level.

Microscopes4Schools is a hands-on science outreach activity for primary school children, now led by Mathias Pasche and delivered by volunteer scientists from the LMB.

They visit local schools to provide a short interactive talk about cells and microscopy, which is followed by a practical hands-on session where pupils can use high-quality educational microscopes to look at different biological samples such as banana cells, water fleas and even their own cheek cells.

Its about giving children an experience that could spark an interest in science, said Simon.

A basic microscope can cost 40, while a higher-quality one will set you back about 300. Budgetary pressures mean that many pupils dont otherwise get to use a microscope until they are in secondary school or sixth-form although parents can inspire their children if they have a microscope at home.

Things that are moving are great for children, said Simon. In summer you can get some pond water and see a lot of life. You can also look at bacteria in yoghurt, or pond weed from an aquarium.

You can find out more about Microscopes4Schools and find valuable resources on experiments on the Microscopes4Schools website.

Go here to read the rest:
How Cambridge scientists are exploring the incredible transport system inside our cells - Cambridge Independent (registration)