Culture

Scientists can now edit multiple sites in the genome at the same time to learn how different DNA stretches co-operate in health and disease.

CRISPR-based DNA editing has revolutionized the study of the human genome by allowing precise deletion of any human gene to glean insights into its function. But one feature remained challenging--the ability to simultaneously remove multiple genes or gene fragments in the same cell. Yet this type of genome surgery is key for scientists to understand how different parts of the genome work together in the contexts of both normal physiology and disease.

Now such a tool exists thanks to the teams of Benjamin Blencowe and Jason Moffat, both professors of molecular genetics at the Donnelly Centre for Cellular and Biomolecular Research. Dubbed 'CHyMErA', for Cas Hybrid for Multiplexed Editing and Screening Applications, the method can be applied to any type of mammalian cell to systematically target the DNA at multiple positions at the same time, as described in a study published in the journal Nature Biotechnology.

Often described as genome scissors, CRISPR works by sending a DNA-cutting enzyme to desired sites in the genome via guide RNA molecules, engineered to adhere to the target site. The most widely used DNA-cutting enzyme is Cas9.

Since Cas9 first came to light, other Cas enzymes with distinct properties have been identified by scientists seeking to improve and expand the applications of the technology. Unlike the CRISPR-Cas9 technology, CHyMErA combines two different DNA-cutting enzymes, Cas9 and Cas12a, to allow more versatile applications. Cas12a is an enzyme that can be used to generate multiple guide RNA molecules in the same cell, which is key for simultaneous DNA editing.

Thomas Gonatopoulos-Pournatzis, a research associate in Blencowe's group, had spent several years trying to develop combinatorial gene editing by testing Cas9 and Cas12a enzymes on their own. He then had the idea to combine these enzymes to generate the CHyMErA system.

"We had been trying a number of approaches to induce genetic fragment deletions and nothing worked as well as CHyMErA," he says. "I was thrilled when together with Shaghayegh Farhangmehr, a PhD student in the Blencowe lab, we saw the first evidence that CHyMErA was successful in deleting gene segments. We obtained these results on Boxing Day and it was the best Christmas present I could have wished for."

The next step was to harness CHyMErA in large-scale screens to systematically analyze how genes act together, as well the functions of individual parts of genes. Blencowe's team, which studies the regulation and function of gene segments known as exons, approached Moffat, whose group had developed extensive experience with CRISPR technology.

"With CHyMErA, you can use the best of the two enzymes," says Michael Aregger, a research associate in the Moffat lab, who played a key role in developing the screen-based applications of CHyMErA. "Cas9 has been improved by the community to have a very high editing efficiency, whereas Cas12a allows multiplexing of guide RNAs and therefore provides a lot more flexibility in finding sites in the genome that we can cut."

In one application of CHyMErA, the researchers targeted pairs of genes known as paralogs, which have a similar DNA code but remain poorly studied because they were difficult to research. Because paralogs arose by duplication of an ancestral gene, it had been assumed they would largely have similar roles. But their function could not be revealed by the existing single-gene targeting methods typically employed in genetic screens, mostly because the other paralog would compensate for the one that's missing.

"With CHyMErA, we can take out both paralogs in pairs to see if that ancestral function is important for the cell to survive," says Kevin Brown, senior research associate in the Moffat lab and co-lead author on the study along with Aregger and Gonatopoulos-Pournatzis. "We are able to now interrogate a class of genes that was previously missed."

After knocking out ~700 paralog pairs, almost all that exist in the human genome, the analysis confirmed that many of these gene pairs do indeed perform similar roles in cell survival, whereas others have distinct functions.

Another feature of CHyMErA is that both Cas9 and Cas12a can be deployed to nearby genome sites to cut out gene fragments such as exons. This allowed the team to individually delete thousands of exons that have been linked to cancer and brain function but were not amenable to targeting with Cas9 alone. Exons are variably included into genes' transcripts and can modify the function of the encoded proteins, although how individual exons contribute to cellular processes remains largely unknown. Out of 2,000 exons analyzed by CHyMErA, over 100 were found to be critical for cell survival, enabling future research to now focus on shining light on their potential roles in disease.

"Once we identify exons that have a critical role in disease, we can use this information to develop new therapies," says Gonatopoulos-Pournatzis.

Males that face tougher competition for females risk having offspring with a greater number of harmful mutations in their genome than males without rivals. Researchers at Uppsala University have discovered this correlation in the beetle species Callosobruchus maculatus. Their study is published in the scientific journal Nature Ecology & Evolution.

"Many researchers working in the fields of human reproductive biology and more general evolutionary theory have taken an interest in this. The hypothesis is not new in itself but there have been few experiments conducted to test it. This is where we hope our study can contribute an important piece of the puzzle," says David Berger of Uppsala University's Department of Ecology and Genetics.

Just as with fish, birds and mammals, in the insect world several males often mate with the same female. This leads to a form of sexual selection in which the males' sperm compete to fertilise the female's eggs. Males that produce more numerous or more competitive sperm often win the competition and become fathers.

Research conducted at the Department of Ecology and Genetics at Uppsala University has succeeded in demonstrating that increased competition between males can lead to a higher rate of harmful mutations in offspring.

Genomic DNA is damaged with every cell division but this damage is usually prevented or repaired by an effective, but costly, cellular surveillance system. The new study shows that sperm production in competing males of the species Callosobruchus maculatus, or cowpea weevil, comes at the expense of this cellular surveillance.

In experiments, male beetles were exposed to radiation in order to damage their genome. After a period of recuperation, the males were allowed to mate with females and become fathers. The researchers then followed their offspring to measure the varying quality of subsequent generations and discovered that males kept in groups, with the concomitant risk of sperm competition, had offspring with a greater number of harmful new mutations than those that lived alone.

The researchers behind the study do however point out that competition between males need not lead to deteriorating gene health in the long term. This is because, as the study also shows, males from populations with high sperm competition over many generations adapt to the new conditions by producing more sperm and more viable offspring compared to males adapted to a life of monogamy.

"Even if the direct effect of sperm competition is to increase the number of mutations in offspring, the paradoxical long-term effect of sexual selection may be a lower rate of mutation," explains David Berger.

The researchers behind the study explain that both of these mechanisms play important roles in how genetic variation arises and is maintained in species where males compete to mate. This in turn can affect the potential for evolutionary adaptation, which depends on genetic variation.

To eat or not to eat fish is a question that has long concerned pregnant women. Now, a new USC study shows that children whose mothers ate fish from one to three times a week during pregnancy were more likely to have a better metabolic profile -- despite the risk of exposure to mercury -- than children whose mothers ate fish rarely (less than once a week).

The findings appear today in JAMA Network Open.

"Fish is an important source of nutrients, and its consumption should not be avoided," said Dr. Leda Chatzi, associate professor of preventive medicine at the Keck School of Medicine of USC and the senior investigator on the study. "But pregnant women should stick to one to three servings of fish a week as recommended, and not eat more, because of the potential contamination of fish by mercury and other persistent organic pollutants."

Fish is a major source of omega-3 long-chain polyunsaturated fatty acids which are important for the developing fetus. However, some types, such as swordfish, shark and mackerel, can contain high levels of mercury - a potent toxin that can cause permanent neurological damage. Mercury contamination is also found in soil, air, water and plants.

Researchers looked at 805 mother and child pairs from five European countries participating in a collaborative research project known as the HELIX study, which is following women and their children from pregnancy onwards.

During their pregnancy, the women were asked about their weekly fish consumption and tested for mercury exposure. When the children were from 6 to 12 years old, they underwent a clinical examination with various measurements including waist circumference, blood pressure, high-density lipoprotein cholesterol, triglyceride levels and insulin levels. These measures were combined to calculate a metabolic syndrome score.

More fish is not better

The children of women who ate fish from one to three times a week had lower metabolic syndrome scores than the children of women who ate fish less than once a week. But the benefit declined if women ate fish more than three times a week.

"Fish can be a common route of exposure to certain chemical pollutants which can exert adverse effects," said Nikos Stratakis, PhD, a USC postdoctoral scholar who was one of the study authors. "It is possible that when women eat fish more than three times a week, that pollutant exposure may counterbalance the beneficial effects of fish consumption seen at lower intake levels."

The study found that higher mercury concentration in a woman's blood was associated with a higher metabolic syndrome score in her child.

The study also examined how fish consumption by the mother affected the levels of cytokines and adipokines in her child. These biomarkers are related to inflammation, a contributor to metabolic syndrome. Compared with low fish intake, moderate and high fish consumption during pregnancy were associated with reduced levels of proinflammatory cytokines and adipokines in the children.

This is the first human study to show that the reduction in these inflammation biomarkers could be the underlying mechanism explaining why maternal fish consumption is associated with improved child metabolic health.

Next, the researchers plan to look at the effects of consuming different types of fish with different nutrients and mercury levels and to follow up on these children until the age of 14-15 years.

NEW YORK (March 16, 2020)—Undetected cases, many of which were likely not severely symptomatic, were largely responsible for the rapid spread of the COVID-19 outbreak in China, according to new research by scientists at Columbia University Mailman School of Public Health. The findings based on a computer model of the outbreak are published online in the journal Science.

The researchers report:

86 percent of all infections were undocumented prior to the January 23 Wuhan travel shutdown

Per person, these undocumented infections were half (52 percent) as contagious as documented infections yet were the source of two-thirds of documented infections

Government control efforts and population awareness have reduced the rate of spread of the virus in China; after travel restrictions and control measures were imposed, it spread less quickly

"The explosion of COVID-19 cases in China was largely driven by individuals with mild, limited, or no symptoms who went undetected," says co-author Jeffrey Shaman, PhD, professor of environmental health sciences at Columbia University Mailman School. "Depending on their contagiousness and numbers, undetected cases can expose a far greater portion of the population to virus than would otherwise occur. We find for COVID-19 in China these undetected infected individuals are numerous and contagious. These stealth transmissions will continue to present a major challenge to the containment of this outbreak going forward."

The researchers used a computer model that draws on observations of reported infection and spread within China in conjunction with mobility data from January 10-23 and January 24-February 8. They caution that major changes to care-seeking or patient documentation practices, as well as rapid developments with regard to travel restrictions and control measures, may make predictions difficult.

"Heightened awareness of the outbreak, increased use of personal protective measures, and travel restriction have helped reduce the overall force of infection; however, it is unclear whether this reduction will be sufficient to fully stem the virus spread," says Shaman. "If the novel coronavirus follows the pattern of 2009 H1N1 pandemic influenza, it will also spread globally and become a fifth endemic coronavirus within the human population."

Acne treatment including the highly effective acne medication isotretinoin should be made more readily available despite reports of its association with depression and teen suicide. That's the implication of a study just published in the Journal of the American Academy of Dermatology.

Isotretinoin has been linked to depression and suicide. But UCR researcher Misaki Natsuaki, one of the study's authors, said severe acne itself, when left untreated, poses a serious risk for depression and anxiety.

Natsuaki's research team conducted the first meta-analysis, analyzing 42 studies involving more than one million people, to resolve sometimes-conflicting data related to studies of how acne is related to depression and anxiety. The studies showed a significant association with acne and mental health.

"Acne may not cause physical pain or impairment, but you do not want to leave acne untreated because that's a risk for mental health," said Natsuaki, an associate professor of psychology and director of the UCR Developmental Transitions Laboratory. "Undertreatment of acne itself is related to depression and anxiety, especially in females."

Isotretinoin, often referred to by the since-discontinued brand name Accutane, is an effective acne medication that hit the market in 1982. It can reduce the new acne after a standard six months treatment by reducing the size of oil glands and the amount of oil they produce, and making pores less hospitable to the Propionibacterium acnes bacteria. In up to 90% of cases, all or nearly all of acne is eliminated. Its brand names include Absorica, Claravis, Amnesteem, Myorisan, and Zenatane.

But in 1998, the Food and Drug Administration issued a warning to doctors regarding a possible association with depression, psychosis, suicidal ideation, and suicide. One estimate - not based on a study - attributed 10 suicides in 2019 to isotretinoin.

The anti-Accutane phenomena, and regulatory action, grew 20 years ago after a congressman attributed his son's suicide to the medication. A subsequent suit was dismissed years later. In 2006, the FDA created the iPledge monitoring program, a risk management system specifically for Isotretinoin. With a goal of enforcing control over prescribing, dispensing, and using isotretinoin, the program requires all prescribers, pharmacists, and patients to register and log detailed information into the site.

Natsuaki's meta-analysis found both depression and anxiety heightened in acne sufferers at an effect size similar to the ill effect of cyberbullying on victim's depression, anxiety, and loneliness. Contrary to expectations, the researchers found that acne is associated with greater anxiety and depression among adults than adolescents.

"We speculate that, although no one likes to have acne on his or her face, the prevalence of acne is high in adolescent years and it's somewhat expected to happen in teens," Natsuaki said. "However, for adults, acne may appear as a deviation from social norm."

The study also found the emotional cost of acne is higher in Middle Eastern regions, though that may have been impacted by the inclusion of a disproportionately high number of studies from countries including Turkey, Iran, and Egypt.

The study concludes: "Given the psychiatric risk of acne itself, it is important for clinicians to optimize acne management which may include utilization of isotretinoin."

In advancing research to tackle the problem of burgeoning global economic inequality, researchers at Simon Fraser University used a poverty simulation game called SPENT to foster greater understanding of what causes poverty and economic inequality.

In research published in the scientific journal Nature Human Behaviour today, a group of researchers, including former SFU PhD student Dylan Wiwad and Lara Aknin, an SFU distinguished professor in the department of psychology, share findings from a five-part global study on people's beliefs about poverty and economic inequality.

Study 1 used pre-existing data from the World Values survey - based on about 30,000 participants - while the researchers collected further data from about 2,400 more participants for the remaining studies.

In all, the studies used data from 34 countries. The last two studies provide some initial evidence that using a relatively easy, cost-effective, and "low-touch" intervention - the 10-minute poverty simulation game called SPENT - can foster lasting and meaningful change in beliefs about poverty and reduce support for economic inequality, says Wiwad, who conducted the study as part of his PhD dissertation at SFU.

Wiwad is currently a post-doctoral scholar at the Kellogg School of Management, Northwestern University.

The researchers invited about 600 students to a laboratory setting to play the game and divided them into two groups. One group was asked to take part in a poverty simulation experience, where they live a month in the life of an impoverished person making daily financial decisions.

In following up with participants over the next few months, the researchers found that playing SPENT helped develop stronger recognition of the situational causes of poverty and lowers support for economic inequality even five months after playing the game.

"How people understand the causes of poverty influences their willingness to address inequality and help the poor," says Aknin.

She adds that the final two studies also show that these interventions are scalable and can be used across classrooms in the province or Canada by teachers.

"Our dream is to partner with the Vancouver School Board and classrooms around the city to investigate if we see similar long-lasting results using these interventions during these impressionable periods."

Some 500 million years ago - when our continents were connected in a single land mass and most life existed underwater - hornworts (Anthoceros) were one of the first groups of plants to colonize land. An international team led by University of Zurich (UZH) and the Boyce Thompson Institute has now sequenced three hornwort genomes, providing insights into the genetics underlying the unique biology of the group, an extant representative of the earliest land plants.

The research team began the project in 2011. "It took us three years to figure out how hornworts can be grown and pushed through its sexual life cycle under laboratory conditions, and another three years to properly assemble and annotate its genome," says Péter Szövényi, researcher at UZH and last author of the paper.

Higher crop yields with less fertilizer

One of the researchers' goals was to find genes that play a role in hornworts' method of concentrating carbon dioxide inside chloroplasts, which boosts the plants' ability to make sugar resulting in increased yield. Hornworts are unique among land plants in this capability, but some species of algae share the trait. The researchers thus compared the hornwort genomes with those of algae and found one gene, LCIB, that is shared by the two groups of plants but not with other land plants. "If this carbon-concentrating mechanism could be installed in crop plants, then they could grow larger with the same amount of fertilizer," explains first author Fay-Wei Li, plant biologist at the Boyce Thompson Institute and Cornell University.

Symbiosis with bacteria to acquire nitrogen

Furthermore, hornworts live in symbiosis with fungi and cyanobacteria providing phosphorus and nitrogen to the plant. The researchers also identified 40 genes that may promote the hornworts' source of nitrogen, which comes from an interdependent relationship with cyanobacteria - a unique feature in land plants. "If this capability of hornworts can be transferred to crop plants, many tons of nitrogen fertilizer could be saved," says Szövényi. Such a reduction in fertilizer could benefit the environment, since excess agricultural nitrogen frequently enters waterways, where it can cause deadly algal blooms. Szövényi and Li are already working on a project to understand the genetic mechanism underlying the symbiotic plant-cyanobacteria interaction.

Illuminating the origin of land plants

The research also shed light on the evolution of early land plants. Without stomata, most plants cannot take up carbon dioxide and thrive in the terrestrial environment. Therefore, stomata represent a key innovation in colonization of the terrestrial environment. Nevertheless, until now it was unknown whether stomata have evolved once or potentially multiple times independently in land plants. Hornworts possess stomata during their spore-producing phase. "We found that the basic genetic toolkit governing stomatal development in flowering plants is shared with hornworts," explains Szövényi. This finding is consistent with the hypothesis that stomata have evolved only once in the most recent common ancestor of land plants.

Hornworts, liverworts and mosses were among the first plants to colonize land, but how the three groups were related had previously not been clear. "Our research shows clearly that hornworts, liverworts and mosses are all more closely related to each other than they are to vascular plants. We also show that liverworts and mosses are more closely related to each other than to hornworts," says Fay-Wei Li.

DNA only persists through replication - naturally or synthetically. While humans need the genetic material to be reproduced in order to replace old or damaged cells, the ability to replicate DNA in a laboratory setting can provide researchers insights into the mechanisms of disease or the platform to develop treatments.  

Moreover, large-scale synthesis of DNA via such replication is a cornerstone of synthetic biology. However, just like the way we write or type an article, this DNA "writing" process is error prone, which has become a serious problem in large-scale DNA synthesis.

Now, a team of researchers from the Chinese Academy of Science (CAS) has developed a more efficient and cost-effective way to accurately synthesize DNA than traditionally used methods. They published their results on March 5, 2020 in ACS Synthetic Biology.  

"In synthetic biology, genes, gene networks and even entire genomes are synthesized to create new functions," said ZHANG Jia, paper first-author and a researcher from Single-Cell Center, Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT), CAS.  

The synthetization process involves assembling DNA in liquid or on a microchip outfitted to intentionally pair specific genetic fragments. The problem, according to ZHANG, is that the fragments frequently mismatch and produce significant errors. Current methods to reduce these errors involve a protein called MutS, which is attracted to mismatched genetic fragments. The protein acts as a flag on the errors, allowing scientists to identify and remove them.  

This process is costly and time-consuming, however. One major reason is that, the key error-correcting enzyme of eMutS, a protein derived from E. coli and binds errors with high accuracy, is fragile and does not last long.  

"To tackle this challenge, we have developed a simple, effective and cost-efficient error-correction system that is readily applicable in gene synthesis workflow," ZHANG said. 

The CAS team began by treating eMutS with chemical stabilizers in the form of a kind of molecular glue called disulfide bonds. With a strong chemical structure, the introduced bonds, plus improvements in enzyme production and storage, extended the life of the proteins from seven to 63 days. Preparing the proteins for the error-removal process can take a significant amount of time, so this remarkable increase in enzyme durability means that researchers can go from preparing proteins once a week to once every two months. In industry-scale DNA synthesis workflows, this means significant reduction in operation, labor and time costs. 

Furthermore, using the newly durable eMutS protein,  86.4% of the synthetic DNA fragments are completely free of errors - a nearly seven-fold increase in accuracy from the commercial enzyme systems currently on the market, according to ZHANG.  

"This system's high fidelity, simple operation and low cost in error correction address one of the key challenges in DNA synthesis and could have implications for broad applications in synthetic biology, including industrial applications," said XU Jian, senior author of the study and Director of Single-Cell Center, QIBEBT, CAS.  

The team plans to continue improving the shelf-life of the proteins while also further increasing the accuracy of error removal in DNA synthesis.  

Silkworms are useful for more than just making strong and absorbent strings of silky thread for the textiles industry. A group of KAUST scientists has now coaxed these grub-like insects into making the human form of E-selectin, a critical adhesion molecule involved in inflammation, cancer and other disease processes.

Working with collaborators at Kyushu University in Japan, biochemist Jasmeen Merzaban and her team engineered silkworms to produce different variants of E-selectin. They then studied how the various adhesion proteins interacted with cells, discovering that there is more to E-selectin's molecular stickiness than how its terminal binding domain interacts with target molecules.

For the first time, they have shown that critical for binding is the connecting arm of E-selectin --a repetitive region of variable length (known as the short consensus repeat domain) that extends the protein's hand to grab cells out of flow.

Longer armed E-selectins are better at tethering blood stem cells, Merzaban and her colleagues report. But the speed with which E-selectin grabs the cells seems controlled only by the protein's hand and wrist--the lectin and the epidermal growth-factor-like domains, respectively.

The findings shed light on the natural trafficking of blood components into and out of tissues. They might also explain how rogue cancer cells in the blood get captured at distant organs to seed new tumors throughout the body.

"Evaluating the ability of components of the E-selectin molecule to block adhesion interactions based on our work could generate results with therapeutic implications," Merzaban says.

She and members of her are currently exploring the potential for lab-grown E-selectin proteins to serve as decoys in the body. These molecules should get between cancer cells and natural E-selectins found in blood vessels or in the bone marrow, and thereby reduce the rate of metastasis, the main cause of cancer-related death, or prevent tumor cells from hiding away in organs where they are protected from chemotherapy.

To manufacture more E-selectin, the team will again rear huge numbers of transgenic creamy white silkworms. Other options for large-scale protein production abound, including the use of Chinese hamster ovary cells and bacterial culture systems. But rodent cells can be expensive and inefficient in making recombinant proteins and bacteria cannot always faithfully replicate aspects of mammalian protein biology.

"The silkworm expression system presents the advantage of producing functional mammalian proteins at large scale with high yields at low cost," says Fajr Aleisa, the first author of the study and a former Ph.D. student in Merzaban's lab group.

Eating more protein at breakfast or lunchtime could help older people maintain muscle mass with advancing age - but most people eat proteins fairly unevenly throughout the day, new research at the University of Birmingham has found.

The body's mechanisms for producing new muscle require regular stimulation to function efficiently - this stimulation happens when we eat protein. The mechanisms are less efficient in older people, so they need to eat more protein to get the same response as younger people.

But just eating more protein is not enough, though - older people also need to spread that intake evenly across all their meals to ensure they maximise the benefits of protein for muscle mass.

Researchers in the School of Sport, Exercise and Rehabilitation Sciences at the University of Birmingham, studied the dietary intake of young, middle-aged and old-aged individuals with a particular focus on the amount, pattern and source of protein consumed.

Their results showed that, while the majority of individuals across all three groups met or exceeded current national guidelines (RDA) for protein intake, the protein intake and distribution across daily meals and snacks were very varied.

The study involved 120 participants divided into three age groups. In the first, participants had an average age of 23; in the second an average age of 51; and in the third an average age of 77. All participants were asked to complete a food diary over a three-day period, weighing out every single food item consumed.

Researchers looked for patterns in the dietary behaviour of participants. In particular, they evaluated the protein intake across the different age groups and found 18 different patterns of protein intake throughout the day, showing a wide variety of eating habits.

Most noticeably, the team found that old people, compared to young and middle-aged individuals, people were more likely to eat a lower-quality protein source, such as bread, at lunchtime.

The results offer compelling evidence for revised nutritional guidelines that could help older people adopt habits that spread consumption of good quality proteins across all their meals.

"We know that older people show a blunted response to muscle building when consuming a certain amount of protein. Therefore, older individuals need to eat more protein to get the same muscle building response as younger and middle-aged people," explains Dr Benoit Smeuninx, first author on the study. "another way to help muscles make better use of dietary protein is to perform regular exercise".

"Most people are reaching the Recommended Daily Allowance of protein, but our results show that a one-size-fits-all guideline for protein intake isn't appropriate across all age groups. Simply saying older people should eat more protein isn't really enough either. We need a more sophisticated and individualised approach that can help people understand when and how much protein to consume to support muscle mass."

Future areas for research include studying how protein needs in hospitalised individuals could aid the maintenance of muscle mass, and to further elucidate the interaction between physical activity protein consumption in the fight against age-related muscle loss.

Many genetic and breeding studies have shown that point mutations and indels (insertions and deletions) can alter elite traits in crop plants. Although nuclease-initiated homology-directed repair (HDR) can generate such changes, it is limited by its low efficiency. Base editors are robust tools for creating base transitions, but not transversions, insertions or deletions. Thus, there is a pressing need for new genome engineering approaches in plants.

David R. Liu and his colleagues at Harvard University developed a new genome editing approach, prime editing, which uses engineered Cas9 nickase (H840A)-reverse transcriptase (RT) fusion proteins paired with a prime editing guide RNA (pegRNA) that encodes the desired edit in human cells.

Recently, a research team led by Prof. GAO Caixia of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences reported the optimization of a prime editing system (PPE system) for creating desired point mutations, insertions and deletions in two major cereal crops, namely, rice and wheat. The main components of a PPE system are a Cas9 nickase-RT fusion protein and a pegRNA.

Using the PPE system, these researchers produced all 12 kinds of single base substitutions, as well as multiple point mutations and small DNA insertions and deletions at 9 rice and seven wheat sites in protoplasts, with efficiencies up to 19.2%. The efficiency of PPE was strongly affected by the length of the primer binding site (PBS) and RT template.

Although byproducts (off-target effects) were generated by the PPE system, they can be reduced by optimizing RT template length. Moreover, using a PPE system optimized for plants, they found that the original RT could be replaced by CaMV-RT (from the cauliflower mosaic virus) and retron-derived RT (from E. coli BL21). Prime editing efficiency was also improved at some targets by using their PPE-Ribozyme (PPE-R) and by incubating at 37 ?.

Furthermore, GAO and her collaborators were able to create stable mutant rice plants carrying G-to-T point mutations, multinucleotide substitutions, and a number of desired 6-nt deletions, with a mutant production efficiency approaching 22%. It is noteworthy that these three types of mutation are very difficult to produce with current editing tools.

"Although the efficiency of the PPE system is lower than that of base editors, it is still an appealing new tool for creating all 12 types of single-point mutation, mixtures of different substitutions, and insertions and deletions. The system thus has great potential for plant breeding and functional genomics research," said Dr. GAO.

Advances in materials, microfabrication and medical imaging are accelerating the pace of innovation in implantable neuroprosthetics. These soft, biocompatible devices, which rely on the electrical stimulation of the nervous system, have shown enormous potential in improving quality of life for patients with a range of conditions such as paralysis and deafness. Despite their promise, most of these groundbreaking discoveries never make it out of the lab.

Few discoveries make it to market

Researchers tend to underestimate the amount of engineering and development work needed to get a discovery out of the lab and into clinical use. "These discoveries can be so far ahead of the curve that finding an industry partner willing to do the development work is practically impossible," says Stéphanie Lacour, who heads EPFL's Laboratory for Soft Bioelectronic Interfaces (LSBI).

There is often a disconnect between the evaluation of new implantable devices and the physical and biological reality of healthcare practice. "With a new soft, implantable interface, you have to go through extra validation steps that don't apply to conventional implants," adds Lacour.

The EPFL team has developed an experimental protocol for testing, optimizing and validating soft, personalized implants such as the devices developed by researchers at the LSBI. The method, which should help facilitate the transition from lab to market, is described in a paper published in the journal Advanced Materials.

A four-step process

The first step is to develop a personalized, anatomically accurate, biophysical prototype of the tissue into which the device will be implanted. This process is made possible by medical imaging data and 3D printing techniques. "This means that, for each patient, we reproduce the precise structure of the tissue that will ultimately host the implant," explains Giuseppe Schiavone, a scientist at the LSBI.

Next, the fabrication process is optimized to make sure it is both reliable and reproducible. The soft implant is inserted into the tissue prototype, which is then placed in an environment that replicates the biophysical conditions inside the human body. Using a biomimetic platform developed in the lab, the researchers apply mechanical stimuli to the implant and the surrounding tissue to mimic the dynamic in vivo environment and to validate the device's biocompatibility and therapeutic efficacy. The platform also ages the implant physically and electrochemically under near-realistic conditions.

Fine-tuning through tests

"With this method, we're able to test implants more quickly, realistically and cheaply without the need for surgical procedures," adds Schiavone. "And because we evaluate the device at each stage of the process, we can fine-tune and improve it on the fly with minimal disruption." According to Schiavone, a lack of standardized validation processes for soft, biocompatible implants means that research teams currently devise their own protocols.

Researchers at the LSBI worked with the group of Marco Capogrosso at the University of Fribourg and EPFL and Lausanne University Hospital (CHUV) teams led by Grégoire Courtine and Jocelyne Bloch to test the protocol on e-dura, an EPFL-developed neuromodulation device implanted in the epidural space of the spinal cord.

Although these new soft, implantable devices are not yet ready to proceed to clinical trial, the paper's authors hope that their work will encourage other researchers to apply translational principles and support further advances in medtech innovation.

CHAMPAIGN, Ill. -- Two brain-signaling molecules control how anemonefish dads care for their young and respond to nest intruders, researchers report in a new study. Because there are many similarities in brain structure between fish and humans, the findings offer insight into the fundamental nature of parental care, the scientists say.

"One of the benefits of studying fish is that their behaviors are simpler," said Ross DeAngelis, a former graduate student who conducted the work in the laboratory of Justin Rhodes, a professor of psychology at the University of Illinois at Urbana-Champaign. "By exploring these systems, we can understand the broader implications on vertebrate behavior."

Anemonefish live in pairs on sea anemones in the wild. Female anemonefish are more aggressive than their mates and help protect the nest from intruders. Fathers have a more nurturing role. See video.

"Male anemonefish are spectacular fathers," said Rhodes, who led the research. "They nourish the eggs by fanning them to provide oxygen and clear debris, and they nip at the eggs to clean them. When a predator is around, they switch their behavior to become aggressive - they try to bite and fight the predators away."

Previous research focused on only one facet of parental care: either nourishment or defense. The new study aims to understand both aspects together in the presence of intruders.

Two hormones play a role in guiding the trade-off between caring for one's offspring and defending them. Arginine vasotocin increases aggression, while isotocin boosts egg care. Their effects on parental behavior in the presence of intruders was previously unknown.

The researchers used inhibitory compounds, known as antagonists, to block the binding of arginine vasotocin or isotocin to their receptors in the brain. They injected these compounds into the abdomens of the fish. From there, the antagonists were carried to the brain through the blood.

"Blocking arginine vasotocin reduced aggression and increased parental care in male anemonefish," DeAngelis said. "This is an unusual result because they are such good dads - we didn't think it was possible for them to be even better."

Blocking isotocin had the opposite effect. It increased aggression, and the fish spent less time nipping and fanning their eggs, DeAngelis said.

"The results are similar to what we see in humans," Rhodes said. "Oxytocin, which is the human version of isotocin, is known to be important for nurturing. Arginine vasopressin, which is the human version of arginine vasotocin, plays a role in social and affiliative behavior in the slightly different context of mating."

The two hormones have very similar structures and bind to similar receptors in the brain, so the researchers cannot be certain that the antagonist for one isn't also affecting the other. They also have not yet determined how the hormones specifically modify brain-signaling.

"Individuals across the animal kingdom have to make decisions on how to maximize their fitness, and most of those decisions are based on environmental context," DeAngelis said. "It is interesting to see that the neurochemical pathways can be modulated by the current social context."

Scientists from the University of Sheffield have discovered a pulsating ancient star in a double star system, which will allow them to access important information on the history of how stars like our Sun evolve and eventually die.

The discovery of the first ever pulsating white dwarf star in an eclipsing binary by physicists at Sheffield means the team can see how binary evolution has affected the internal structure of a white dwarf in detail for the first time.

An eclipsing binary, or double star system, is made up of two stars orbiting each other and periodically passing in front of each other as seen from the Earth.

White dwarf stars are the burnt out cores left behind when a star like the Sun dies. This particular white dwarf could provide key insights into the structure, evolution and death of these stars for the first time.

Most white dwarfs are thought to be made primarily of carbon and oxygen, but this particular white dwarf is made mostly of helium. The team think this is a result of its binary companion cutting off its evolution early, before it got a chance to fuse the helium into carbon and oxygen.

The pulsations from this star were discovered using HiPERCAM, a revolutionary high-speed camera developed by a team led by Professor Vik Dhillon from the University of Sheffield's Department of Physics and Astronomy.

HiPERCAM can take one picture every millisecond simultaneously in five different colours and is mounted on the 10.4m Gran Telescopio Canarias (GTC), the world's largest optical telescope on La Palma. This allowed the researchers to detect the rapid and subtle pulsations from this particular white dwarf.

The pulsations of the white dwarf star and the eclipsing binary system allowed the team to investigate the structure of it using two techniques, asteroseismology and eclipse studies.
Asteroseismology involves measuring how fast sound waves travel through the white dwarf.

Dr Steven Parsons, from the University of Sheffield's Department of Physics and Astronomy, who led the study said: "Determining what a white dwarf is made of is not straightforward because these objects have about half of the mass of the Sun, packed into something about the size of the Earth. This means that gravity is extremely strong on a white dwarf, around one million times larger than here on Earth, so on the surface of a white dwarf an average person would weigh about 60,000,000kg. The gravity causes all of the heavy elements in the white dwarf to sink to the centre, leaving only the lightest elements at the surface and so the true composition of it remains hidden underneath.

"This pulsating white dwarf we discovered is extremely important since we can use the binary motion and the eclipse to independently measure the mass and radius of this white dwarf, which helps us determine what it is made of. Even more interestingly, the two stars in this binary system have interacted with each other in the past, transferring material back and forth between them. We can see how this binary evolution has affected the internal structure of the white dwarf, something that we've not been able to do before for these kinds of binary systems."

The next step of the research is to continue observing the white dwarf to record as many pulsations as possible using HiPERCAM and the Hubble Space Telescope.

A good indicator of dysregulation in live cells is a change in their RNA expression. MicroRNA (miRNA), a special type of RNA, is considered a biomarker for carcinogenic cells. A team of scientists from China has found a way to amplify miRNA in live tumor cells for bioimaging. As they report in the journal Angewandte Chemie, their assay is based on a robust cellular autocatalytic biocircuit triggered by synthetic DNA and nanoparticles.

Diagnosing cancer before a tumor becomes visible has been one of the long-standing goals in medicine. One of the biomarkers for carcinogenicity in a cell is its RNA expression pattern or, more precisely, the change in RNA expression, which causes metabolic degeneration. There are many types of RNA, among which a short noncoding RNA called miRNA promotes or impedes the translation of nucleus-encoded genetic information into protein. Accordingly, the detection of a changed miRNA expression profile is thought to be a reliable indication of the degeneration of a cell.

However, the detection of a particular miRNA is difficult because it is present in the cell only in tiny amounts and must by amplified and connected to a signaling entity, such as a fluorescence dye, for visualization. A team of scientists at Wuhan University, China, led by Fuan Wang, have discovered a suitable amplification-detection mechanism for miRNA, which relies on an autocatalytic biocircuit activated by synthetic DNA, leading to a strong fluorescence signal that flags tumor cells.

RNA is usually synthesized in the nucleus of the cell and transported to the cytoplasm where it conveys genetic information. However, when synthetic DNA is present in the cytoplasm, RNA can bind to a matching nucleotide sequence of the DNA strand; a fact that is exploited in, for example, antiretroviral treatment to silence viral RNA expression. Wang and his coworkers did the opposite. By matching synthetic DNA strands with miRNA, they triggered an autocatalytic amplification circuit--called autocatalytic DNAzyme biocircuit--to form DNA-miRNA assemblies. These assemblies grew further to form DNAzyme nanowires that carry the fluorescence dyes.

After administering the DNAzyme detection kit, the authors observed bright fluorescence in a mouse model at the location where a tumor was developing.

To make the DNAzyme enter the tumor cells, the authors used nanoparticles--tiny parcels that can deliver drugs and other molecular freight to the cells--made of manganese dioxide with a honeycomb-like structure. According to the authors, this composition and architecture has the advantage that the nanoparticle can be readily activated by glutathione, which is a chemical found in abundance in tumor cells. Another advantage is that the released manganese ions would sustain the autocatalytic DNAzyme biocircuit, the authors write.

The scientists emphasize that their self-enhanced bioimaging system could be developed as a powerful tool to visualize tumor cells with biomarkers. This is especially promising as many different miRNAs can be selectively targeted to investigate different cancers or other cell dysfunction.