Visualizing DNA double-strand break process for the first time

The enzyme I-DmoI (purple) is specifically associated to the double strand of DNA (yellow and green). Credit: CNIO

Scientists from the Spanish National Cancer Research Centre (CNIO), led by Guillermo Montoya, have developed a method for producing biological crystals that has allowed scientists to observe --for the first time-- DNA double chain breaks. They have also developed a computer simulation that makes this process, which lasts in the order of millionths of a second, visible to the human eye. The study is published today by the journal Nature Structural & Molecular Biology.

"We knew that enzymes, or proteins, endonucleases, are responsible for these double strand breaks, but we didn't know exactly how it worked until now," said Montoya. "In our study, we describe in detail the dynamics of this basic biological reaction mediated by the enzyme I-Dmol. Our observations can be extrapolated to many other families of endonucleases that behave identically."

DNA breaks occur in several natural processes that are vital for life: mutagenesis, synthesis, recombination and repair. In the molecular biology field, they can also be generated synthetically. Once the exact mechanism that produces these breaks has been uncovered, this knowledge can be used in multiple biotechnological applications: from the correction of mutations to treat rare and genetic diseases, to the development of genetically modified organisms.

Slow-motion reaction

Enzymes are highly specialised dynamic systems. Their nicking function could be compared, said Montoya, to a specially designed fabric-cutting machine that "it would only make a cut when a piece of clothing with a specific combination of colours passed under the blade."

In this case, researchers concentrated on observing the conformational changes that occurred in the I-Dmol active site; the area that contains the amino acids that act as a blade and produces DNA breaks.

By altering the temperature and pH balance, the CNIO team has managed to delay a chemical reaction that typically occurs in microseconds by up to ten days. Under those conditions, they have created a slow-motion film of the whole process.

"By introducing a magnesium cation we were able to trigger the enzyme reaction and subsequently to produce biological crystals and freeze them at -200ºC," said Montoya. "In that way, we were able to collect up to 185 crystal structures that represent all of the conformational changes taking place at each step of the reaction."

Finally, using computational analysis, the researchers illustrated the seven intermediate stages of the DNA chain separation process. "It is very exciting, because the elucidation of this mechanism will give us the information we need to redesign these enzymes and provide precise molecular scissors, which are essential tools for modifying the genome," he concluded.

Source: Centro Nacional de Investigaciones Oncologicas (CNIO)

On a safari through the genome: Genes offer new insights into the distribution of giraffes

Three young, male Angola giraffes. Credit: © Julian Fennessy, GCF

The Giraffe (Giraffa camelopardalis), a symbol of the African savanna and a fixed item on every safari's agenda, is a fascinating animal. However, contrary to many of the continent's other wild animals, these long-necked giants are still rather poorly studied. Based on their markings, distribution and genome, nine subspecies are recognized -- including the two subspecies Angola Giraffe (Giraffa c. angolensis) and South African Giraffe (Giraffa c. giraffa).

South African Giraffes occur farther north than previously assumed

Like most other giraffes, these subspecies are now mainly found in nature reserves. Until recently, scientists assumed a clear demarcation of their ranges: Angola Giraffes occur in Namibia and northern Botswana, while South African Giraffes reside in southern Botswana and South Africa. "However, according to our studies, the distribution areas prove to be much more complex. South African Giraffes also occur in northeastern Namibia and northern Botswana, and Angola Giraffes can be found in northwestern Namibia and southern Botswana, as well," explains the study's author, Friederike Bock from the Biodiversity and Climate Research Center (BiK-F). A look at the new distribution map reveals the presence of a population of Angola Giraffes in the Central Kalahari Game Reserve, the world's second-largest national park, quasi nestled between two populations of the South African Giraffe, with both subspecies living side by side.

Subspecies were the result of early geographic separation

According to the research team, the fact that two genetically distinct subspecies could develop within the same region may be explained by the local geographic conditions that prevailed approximately 500,000 to two million years ago. Back then, the mountain range along the East African Rift Valley was sinking, creating vast wetlands and lakes, such as the paleo lake Makgadikgadi. According to Professor Dr. Axel Janke from the BiK-F, "these large bodies of water may have separated the populations for long periods of time. Moreover, female giraffes likely do not migrate across long distances, thereby contributing to a clear separation of the maternal lines." Today, there no longer exist any barriers that prevent the possible mingling of both subspecies; an investigation of these processes is however subject to further genetic analyses.

Angola and South African Giraffes can be uniquely identified by their maternal gene profile

For the study, the researchers created a profile of the subspecies' mitochondrial DNA, using tissue samples from about 160 giraffes from various populations across the entire African continent. On the basis of this genetic material, inherited from the maternal side, the often similarly marked subspecies can be uniquely identified genetically and the relationships between various populations can be clearly demonstrated. "Our focus was on giraffes in southern Africa, in particular in Botswana and South Africa. There, we sampled populations that had not been genetically analyzed before," says Bock.

New insights enable improved protection measures for the giraffe

According to estimates by the World Conservation Organization IUCN, the world's giraffe population is about 100,000 individuals -- showing a decreasing trend. In Botswana alone, the population has dwindled by more than half in recent years. In order to achieve effective protection measures that will preserve the majority of the giraffe's subspecies, it is indispensable to gain knowledge that allows their reliable identification as well as detailed information regarding their distribution. The surprising results concerning the distribution of the two subspecies in Namibia and Botswana emphasize the importance of additional taxonomic research on all giraffe subspecies.

Source: Senckenberg Research Institute and Natural History Museum

Controlling genes with your thoughts

Thoughts control a near-infrared LED, which starts the production of a molecule in a reaction chamber. Credit: Martin Fussenegger et al., Copyright ETH Zurich

It sounds like something from the scene in Star Wars where Master Yoda instructs the young Luke Skywalker to use the force to release his stricken X-Wing from the swamp: Marc Folcher and other researchers from the group led by Martin Fussenegger, Professor of Biotechnology and Bioengineering at the Department of Biosystems (D-BSSE) in Basel, have developed a novel gene regulation method that enables thought-specific brainwaves to control the conversion of genes into proteins -- called gene expression in technical terms.

"For the first time, we have been able to tap into human brainwaves, transfer them wirelessly to a gene network and regulate the expression of a gene depending on the type of thought. Being able to control gene expression via the power of thought is a dream that we've been chasing for over a decade," says Fussenegger.

A source of inspiration for the new thought-controlled gene regulation system was the game Mindflex, where the player wears a special headset with a sensor on the forehead that records brainwaves. The registered electroencephalogram (EEG) is then transferred into the playing environment. The EEG controls a fan that enables a small ball to be thought-guided through an obstacle course.

Wireless Transmission to Implant

The system, which the Basel-based bioengineers recently presented in the journal Nature Communications, also makes use of an EEG headset. The recorded brainwaves are analysed and wirelessly transmitted via Bluetooth to a controller, which in turn controls a field generator that generates an electromagnetic field; this supplies an implant with an induction current.

A light then literally goes on in the implant: an integrated LED lamp that emits light in the near-infrared range turns on and illuminates a culture chamber containing genetically modified cells. When the near-infrared light illuminates the cells, they start to produce the desired protein.

Thoughts Control Protein Quantity

The implant was initially tested in cell cultures and mice, and controlled by the thoughts of various test subjects. The researchers used SEAP for the tests, an easy-to-detect human model protein which diffuses from the culture chamber of the implant into the mouse's bloodstream.

To regulate the quantity of released protein, the test subjects were categorised according to three states of mind: bio-feedback, meditation and concentration. Test subjects who played Minecraft on the computer, i.e. who were concentrating, induced average SEAP values in the bloodstream of the mice. When completely relaxed (meditation), the researchers recorded very high SEAP values in the test animals. For bio-feedback, the test subjects observed the LED light of the implant in the body of the mouse and were able to consciously switch the LED light on or off via the visual feedback. This in turn was reflected by the varying amounts of SEAP in the bloodstream of the mice.

New Light-sensitive Gene Construct

"Controlling genes in this way is completely new and is unique in its simplicity," explains Fussenegger. The light-sensitive optogenetic module that reacts to near-infrared light is a particular advancement. The light shines on a modified light-sensitive protein within the gene-modified cells and triggers an artificial signal cascade, resulting in the production of SEAP. Near-infrared light was used because it is generally not harmful to human cells, can penetrate deep into the tissue and enables the function of the implant to be visually tracked.

The system functions efficiently and effectively in the human-cell culture and human-mouse system. Fussenegger hopes that a thought-controlled implant could one day help to combat neurological diseases, such as chronic headaches, back pain and epilepsy, by detecting specific brainwaves at an early stage and triggering and controlling the creation of certain agents in the implant at exactly the right time.

Source: ETH Zurich

Making lab-grown tissues stronger

Connective tissues like cartilage are made of cross-linked bundles of collagen fibers. UC Davis biomedical engineers have discovered that reducing oxygen or adding an enzyme called LOX can make these bundles stronger. The technique can be used to strengthen both natural cartilage kept in the lab for transplant, and artificial cartilage grown in culture. Credit: Eleftherios Makris and Kyriacos Athanasiou, UC Davis

Lab-grown tissues could one day provide new treatments for injuries and damage to the joints, including articular cartilage, tendons and ligaments.

Cartilage, for example, is a hard material that caps the ends of bones and allows joints to work smoothly. UC Davis biomedical engineers, exploring ways to toughen up engineered cartilage and keep natural tissues strong outside the body, report new developments this week in the journal Proceedings of the National Academy of Sciences.

"The problem with engineered tissue is that the mechanical properties are far from those of native tissue," said Eleftherios Makris, a postdoctoral researcher at the UC Davis Department of Biomedical Engineering and first author on the paper. Makris is working under the supervision of Professor Kyriacos A. Athanasiou, a distinguished professor of biomedical engineering and orthopedic surgery, and chair of the Department of Biomedical Engineering.

While engineered cartilage has yet to be tested or approved for use in humans, a current method for treating serious joint problems is with transplants of native cartilage. But it is well known that this method is not sufficient as a long-term clinical solution, Makris said.
The major component of cartilage is a protein called collagen, which also provides strength and flexibility to the majority of our tissues, including ligaments, tendons, skin and bones. Collagen is produced by the cells and made up of long fibers that can be cross-linked together.

Engineering new cartilage

Researchers in the Athanasiou group have been maintaining native cartilage in the lab and culturing cartilage cells, or chondrocytes, to produce engineered cartilage.

"In engineered tissues the cells produce initially an immature matrix, and the maturation process makes it tougher," Makris said.

Knee joints are normally low in oxygen, so the researchers looked at the effect of depriving native or engineered cartilage of oxygen. In both cases, low oxygen led to more cross-linking and stronger material. They also found that an enzyme called lysyl oxidase, which is triggered by low oxygen levels, promoted cross-linking and made the material stronger.

"The ramifications of the work presented in the PNAS paper are tremendous with respect to tissue grafts used in surgery, as well as new tissues fabricated using the principles of tissue engineering," Athanasiou said. Grafts such as cadaveric cartilage, tendons or ligaments -- notorious for losing their mechanical characteristics in storage -- can now be treated with the processes developed at UC Davis to make them stronger and fully functional, he said.
Athanasiou also envisions that many tissue engineering methods will now be altered to take advantage of this strengthening technique.

Source: University of California - Davis

Genetically identical ants help unlock the secrets of larval fate

Cerapachys biroi ants, native to Asia and introduced globally on tropical and subtropical islands, have no queens and have minimal genetic variation, making them ideal for research on social behavior. Credit: Image courtesy of Rockefeller University

A young animal's genes are not the only genes that determine its fate. The genetic identity of its caretakers matters too. Researchers suspect the interaction between the two can sway the fate of the young animal, but this complex dynamic is difficult to pin down in lab experiments.

However, social insect researchers have found a solution. Rockefeller University's Daniel Kronauer, head of the Laboratory of Insect Social Evolution, and his colleagues are developing a species of small raider ants as a model organism in order to ask questions about the relationships between genes, social behavior and evolution.

In a pair of recent papers, the researchers first explain the unique, and potentially useful, biology of this 2.5-millimeter-long ant. Then, in work with collaborators at the University of Paris 13, they put it to work exploring the interaction between the larvae and their nursemaids, and the influence on the young ants' reproductive success as adults.

Clonal raider ants, the species Cerapachys biroi, reproduce by cloning, and they live in colonies of as many as a few hundred nearly genetically identical workers. This makes these ants ideal for studies testing how a particular genetic makeup responds to different conditions, the researchers write in Current Biology. With the help of collaborators at BGI China, researchers in Kronauer's lab have sequenced the clonal raider ant's genome. This is an important step toward using the ant in the sorts of experiments conducted for years in traditional model organisms, such as mice and fruit flies.

"We have shown that colony mates are extremely closely related to one another, with all of the individuals in a colony being essentially genetically identical. This gives us precise control in experiments because we don't need to account for individual genetic variation," says Peter Oxley, a postdoc in the laboratory who led work establishing the clonal raider ant as a promising new model organism.

In the second study, one of the first to make use of the clonal raider ant, a team led by Serafino Teseo of the University of Paris 13 used the unique aspects of the ants' biology to test the indirect role genes play in shaping the future identity of larvae and whole colonies by looking at the interaction between larvae and adults. They did so by observing the success of two ant clones, A and B, in pure colonies or mixed together into chimeric colonies. They also swapped broods, so A adults raised B larvae and vice versa.

It turned out that A and B larvae developed differently depending on whether A or B nurses raised them. Left alone, pure A colonies produced the most young after six generations, making them more successful than B. However, in mixed colonies, B did better because its larvae more frequently turned into large adults that specialize in egg-laying rather than smaller, foraging-focused individuals.

The researchers suspect an indirect genetic effect -- specifically, a social influence. To begin to tease apart the dynamic, they had adults from one clone raise larvae from the other. Again, B did better when raised by A nurses than any of the other combinations. The results were published in Nature Communications.

The B colony's strategy of favoring reproduction over foraging when raised by A colony nurses smacks of social parasitism, in which one organism exploits another's social behavior for its own benefit. "This doesn't mean B is a parasite in the making, just that uncoupling the normal interaction between larvae and their nearly identical adult nursemaids reveals the presence of this mechanism," Kronauer says.

The study shows that, in social species, genetic makeup alone does not provide enough information to predict social behavior. Instead, interactions between social partners, such as larvae and their caregivers, are crucial determinants and can lead to surprising outcomes.

Source: Rockefeller University

Tinkering with the Tao of pandas

This image shows a panda eating in China's Wolong Nature Reserve. Pandas habitat choices center around the ready availability of bamboo -- lots of bamboo.
 Credit: Sue Nichols, Michigan State University

Good news on the panda front: Turns out they're not quite as delicate -- and picky -- as thought.

Up until now, information gleaned from 30 years worth of scientific literature suggested that pandas were inflexible about habitat. Those conclusions morphed into conventional wisdom and thus have guided policy in China. But a Michigan State University (MSU) research associate has led a deep dive into aggregate data and emerged with evidence that the endangered animal is more resilient and flexible than previously believed.

Vanessa Hull is a postdoctoral research associate at MSU's Center for Systems Integration and Sustainability (CSIS). She has spent three years stalking giant pandas in China's Wolong Nature Reserve. Given the pandas' elusive nature, Hull had a lot of down time.

So she bided her time plowing through literature on panda habitat selection, discovering inconsistencies and lack of consensus on matters crucial as scientists and policymakers struggle to protect the estimated 1,600 remaining wild giant pandas in the 21,300 square kilometers to which the animals have been relegated.

"Panda habitat selection is a complex process that we are still trying to unravel," said Jianguo "Jack" Liu, Rachel Carson Chair in Sustainability and CSIS director. "Pandas are a part of coupled human and natural systems where humans have changed so much in their habitat."

It has been thought pandas demanded a forest with fairly gentle slope (easier to mosey around in while seeking bamboo) at a certain elevation in original, old forest, an abundance of bamboo, and plenty of distance from people. These recommendations, Hull said, come from often-scant research because pandas are difficult animals to study.

"Pandas are difficult to observe and follow in the wild, we're always 10 steps behind them," Hulls said. "We don't know why they're there -- or where they were before and after. There's a lot of guesswork."

Vanessa and her colleagues drew up analysis of all the research projects and sought to separate studies that focus on where pandas live from studies that examine what kind of choices pandas make when multiple types of habitat are available. They discovered that pandas may not be as picky as thought.

The research shows, for instance, that pandas are willing to live in secondary forests -- forests that have been logged and have regrown. They also don't seem as selective about slope, and are willing to climb depending on which of the many varieties of bamboo is growing, or what type of forest it was in. Same for elevation, and the amount of sunshine that hits a piece of panda home.

That's good news. Indications that forests once cut clean by timber harvesting can return to acceptable panda habitat validate current bans on forest harvesting.

They also found that there is a complex relationship between trees and bamboo. Pandas choose different forest types as places to spend their time, as long as bamboo is available.

Hull said consensus would be helpful for future panda habitat research, since the future guarantees change.

"It's exciting to see the flexibility pandas have, or at least see that pandas are choosing areas I didn't think could support them," Hull said. "It gives you hope. They've survived throughout many challenges over so many millions of years, it would be sad to think humans came along and threw it all away. This also suggests we should stay on board and try to make things better for them."

Source: Michigan State University