Unprecedented Control of Genome Editing in Flies Promises Insight Into Human Development, Disease

UW-Madison researchers say fine control of genome editing in fruit flies promises to provide new
insights into embryonic development, nervous system function, and the understanding of human
disease. (Credit: Copyright Jeff Miller)

In an era of widespread genetic sequencing, the ability to edit and alter an organism's DNA is a powerful way to explore the information within and how it guides biological function.

A paper from the University of Wisconsin-Madison in the August issue of the journal Genetics takes genome editing to a new level in fruit flies, demonstrating a remarkable level of fine control and, importantly, the transmission of those engineered genetic changes across generations.

Both features are key for driving the utility and spread of an approach that promises to give researchers new insights into the basic workings of biological systems, including embryonic development, nervous system function, and the understanding of human disease.

"Genome engineering allows you to change gene function in a very targeted way, so you can probe function at a level of detail" that wasn't previously possible, says Melissa Harrison, an assistant professor of biomolecular chemistry in the UW-Madison School of Medicine and Public Health and one of the three senior authors of the new study.

Disrupting individual genes has long been used as a way to study their roles in biological function and disease. The new approach, based on molecules that drive a type of bacterial immune response, provides a technical advance that allows scientists to readily engineer genetic sequences in very detailed ways, including adding or removing short bits of DNA in chosen locations, introducing specific mutations, adding trackable tags, or changing the sequences that regulate when or where a gene is active.

The approach used in the new study, called the CRISPR RNA/Cas9 system, has developed unusually fast. First reported just one year ago by scientists at the Howard Hughes Medical Institute and University of California, Berkeley, it has already been applied to most traditional biological model systems, including yeast, zebrafish, mice, the nematode C. elegans, and human cells. The Wisconsin paper was the first to describe it in fruit flies and to show that the resulting genetic changes could be passed from one generation to the next.

"There was a need in the community to have a technique that you could use to generate targeted mutations," says Jill Wildonger, a UW-Madison assistant professor of biochemistry and another senior author of the paper. "The need was there and this was the technical advance that everyone had been waiting for."

"The reason this has progressed so quickly is that many researchers -- us included -- were working on other, more complicated, approaches to do exactly the same thing when this came out," adds genetics assistant professor Kate O'Connor-Giles, the third senior author. "This is invaluable for anyone wanting to study gene function in any organism and it is also likely to be transferable to the clinical realm and gene therapy."

The CRISPR RNA/Cas9 system directs a DNA-clipping enzyme called Cas9 to snip the DNA at a targeted sequence. This cut then stimulates the cell's existing DNA repair machinery to fill in the break while integrating the desired genetic tweaks. The process can be tailored to edit down to the level of a single base pair -- the rough equivalent of changing a single letter in a document with a word processor.

The broad applicability of the system is aided by a relatively simple design that can be customized through creation of a short RNA sequence to target a specific sequence in the genome to generate the desired changes. Previous genome editing methods have relied on making custom proteins, which is costly and slow.

"This is so readily transferable that it's highly likely to enable gene knockout and other genome modifications in any organism," including those that have not previously been used for laboratory work, says O'Connor-Giles. "It's going to turn non-model organisms into genetic model organisms."

That ease may also pay off in the clinic. "It can be very difficult and time-consuming to generate models to study all the gene variants associated with human diseases," says Wildonger. "With this genome editing approach, if we work in collaboration with a clinician to find [clinically relevant] mutations, we can rapidly translate these into a fruit fly model to see what's happening at the cellular and molecular level."

The work, led by genetics graduate student Scott Gratz, was the joint effort of three UW-Madison labs -- particularly notable, Harrison says, that each is in a different department and headed by a female assistant professor. "This has been an amazing collaboration," she says. "It wouldn't have worked if any one of us had tried it on our own."

A Home for the Microbiome

 Biologists Identify How Beneficial Bacteria Reside and Thrive in Gastrointestinal Tract

A section of mouse colon is shown with gut bacteria (outlined in yellow) residing within the crypt channel. (Credit: Caltech / Mazmanian Lab)

The human body is full of tiny microorganisms -- hundreds to thousands of species of bacteria collectively called the microbiome, which are believed to contribute to a healthy existence. The gastrointestinal (GI) tract -- and the colon in particular -- is home to the largest concentration and highest diversity of bacterial species. But how do these organisms persist and thrive in a system that is constantly in flux due to foods and fluids moving through it? A team led by California Institute of Technology (Caltech) biologist Sarkis Mazmanian believes it has found the answer, at least in one common group of bacteria: a set of genes that promotes stable microbial colonization of the gut.

A study describing the researchers' findings was published as an advance online publication of the journal Natureon August 18.

"By understanding how these microbes colonize, we may someday be able to devise ways to correct for abnormal changes in bacterial communities -- changes that are thought to be connected to disorders like obesity, inflammatory bowel disease and autism," says Mazmanian, a professor of biology at Caltech whose work explores the link between human gut bacteria and health.

The researchers began their study by running a series of experiments to introduce a genus of microbes called Bacteriodesto sterile, or germ-free, mice. Bacteriodes, a group of bacteria that has several dozen species, was chosen because it is one of the most abundant genuses in the human microbiome, can be cultured in the lab (unlike most gut bacteria), and can be genetically modified to introduce specific mutations.

"Bacteriodes are the only genus in the microbiome that fit these three criteria," Mazmanian says.

Lead author S. Melanie Lee (PhD '13), who was an MD/PhD student in Mazmanian's lab at the time of the research, first added a few different species of the bacteria to one mouse to see if they would compete with each other to colonize the gut. They appeared to peacefully coexist. Then, Lee colonized a mouse with one particular species, Bacteroides fragilis, and inoculated the mouse with the same exact species, to see if they would co-colonize the same host. To the researchers' surprise, the newly introduced bacteria could not maintain residence in the mouse's gut, despite the fact that the animal was already populated by the identical species.

"We know that this environment can house hundreds of species, so why the competition within the same species?" Lee says. "There certainly isn't a lack of space or nutrients, but this was an extremely robust and consistent finding when we tried to essentially 'super-colonize' the mice with one species."

To explain the results, Lee and the team developed what they called the "saturable niche hypothesis." The idea is that by saturating a specific habitat, the organism will effectively exclude others of the same species from occupying that niche. It will not, however, prevent other closely related species from colonizing the gut, because they have their own particular niches. A genetic screen revealed a set of previously uncharacterized genes -- a system that the researchers dubbed commensal colonization factors (CCF) -- that were both required and sufficient for species-specific colonization by B. fragilis.

But what exactly is the saturable niche? The colon, after all, is filled with a flowing mass of food, fecal matter and bacteria, which doesn't offer much for organisms to grab onto and occupy.

"Melanie hypothesized that this saturable niche was part of the host tissue" -- that is, of the gut itself -- Mazmanian says. "When she postulated this three to four years ago, it was absolute heresy, because other researchers in the field believed that all bacteria in our intestines lived in the lumen -- the center of the gut -- and made zero contact with the host…our bodies. The rationale behind this thinking was if bacteria did make contact, it would cause some sort of immune response."

Nonetheless, when the researchers used advanced imaging approaches to survey colonic tissue in mice colonized with B. fragilis, they found a small population of microbes living in miniscule pockets -- or crypts -- in the colon. Nestled within the crypts, the bacteria are protected from the constant flow of material that passes through the GI tract. To test whether or not the CCF system regulated bacterial colonization within the crypts, the team injected mutant bacteria -- without the CCF system -- into the colons of sterile mice. Those bacteria were unable to colonize the crypts.

"There is something in that crypt -- and we don't know what it is yet -- that normal B. fragilis can use to get a foothold via the CCF system," Mazmanian explains. "Finding the crypts is a huge advance in the field because it shows that bacteria do physically contact the host. And during all of the experiments that Melanie did, homeostasis, or a steady state, was maintained. So, contrary to popular belief, there was no evidence of inflammation as a result of the bacteria contacting the host. In fact, we believe these crypts are the permanent home ofBacteroides, and perhaps other classes of microbes."

He says that by pinpointing the CCF system as a mechanism for bacterial colonization and resilience, in addition to the discovery of crypts in the colon that are species specific, the current paper has solved longstanding mysteries in the field about how microbes establish and maintain long-term colonization.

"We've studied only a handful of organisms, and though they are numerically abundant, they are clearly not representative of all the organisms in the gut," Lee says. "A lot of those other bacteria don't have CCF genes, so the question now is: Do those organisms somehow rely on interactions with Bacteroides for their own colonization, or their replication rates, or their localization?"

Suspecting that Bacteroides are keystone species -- a necessary factor for building the gut ecosystem -- the researchers next plan to investigate whether or not functional abnormalities, such as the inability to adhere to crypts, could affect the entire microbiome and potentially lead to a diseased state in the body.

"This research highlights the notion that we are not alone. We knew that bacteria are in our gut, but this study shows that specific microbes are very intimately associated with our bodies," Mazmanian says. "They are living in very close proximity to our tissues, and we can't ignore microbial contributions to our biology or our health. They are a part of us."

Funding for the research outlined in the Nature paper, titled "Bacterial colonization factors control specificity and stability of the gut microbiota," was provided by the National Institutes of Health and the Crohn's and Colitis Foundation of America. Additional coauthors were Gregory Donaldson and Silva Boyajian from Caltech and Zbigniew Mikulski and Klaus Ley from the La Jolla Institute for Allergy and Immunology in La Jolla, California.

Source : California Institute of Technology.

By Science and Universe

Altruism or Manipulated Helping? Altruism May Have Origins in Manipulation

Parasitic roundworms cause the abdomen of the Cephalotes atratus ant to turn red like a berry, duping birds into eating it and subsequently spreading the parasite in the birds' droppings. The bright abdomen constitutes a phenotype manipulated by the roundworm. (Credit: Steve Yanoviak)

Manipulation is often thought of as morally repugnant, but it might be responsible for the evolutionary origins of some helpful or altruistic behavior, according to a new study.

In evolutionary biology, manipulation occurs when an individual, the manipulator, alters the behavior of another individual in ways that is beneficial to the manipulator but may be detrimental to the manipulated individual. Manipulation not only occurs in humans and animals but also at the cellular level, such as among cells in a multicellular organism, or in parasites, which can alter the behavior of their hosts.

Consider the case of the parasitic roundworm Myrmeconema neotropicum, which once ingested by the tropical ant Cephalotes atratus in Central and South America, causes the ant to grow a bright red abdomen, mimicking berries. This bright abdomen constitutes a phenotype manipulated by the roundworm. Birds eat the "berries," or infected ants, and then spread the parasite in their droppings, which are subsequently collected by foragingCephalotes atratus and fed to their larva, and the cycle of manipulated behavior begins anew.

In the study published this week in the journal American Naturalist, the researchers developed a mathematical model for the evolution of manipulated behavior and applied it to maternal manipulation in eusocial organisms, such as ants, wasps, and bees, which form colonies with reproductive queens and sterile workers. In the model, mothers produce two broods, and they manipulate the first-brood offspring to stay in the maternal site and help raise the second brood. Mothers can do this by disrupting the offspring's development in some way, for example through poor feeding or aggressive behavior. Manipulated offspring of the first-brood stay and help to raise the second brood. Alternatively, first-brood offspring can resist manipulation and leave.

The researchers show that an offspring's resistance to manipulation may often fail to evolve, if the costs of resistance are high. In a sense, then, helping or altruistic behavior is coerced through manipulation.

"The evidence in so-called primitive eusociality, where helping is often coerced through aggression or differential feeding, appears consistent with these results," said lead author Mauricio Gonzalez-Forero, who conducted the study while a graduate research assistant at the National Institute for Mathematical and Biological Synthesis

Article Source :  National Institute for Mathematical and Biological Synthesis (NIMBioS),

By Science and Universe

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Fresh Analysis of Dinosaur Skulls Shows Three 'Species' Are Actually One

Landmark locations. The locations of the 3D landmarks are presented here in (A) dorsal and (B) lateral views on ZMNH M8137. Since the landmarks were not reflected on either side of the skull, the left lateral landmarks have different landmark numbers than the right lateral landmarks. A 3D model of the skull of ZMNH M8137 is included in Multimedia S1 for reference. Scale = 50 mm. (Credit: Brandon P. Hedrick, Peter Dodson. Lujiatun Psittacosaurids: Understanding Individual and Taphonomic Variation Using 3D Geometric Morphometrics. PLoS ONE, 2013; 8 (8): e69265 DOI: 10.1371/journal.pone.0069265)

A new analysis of dinosaur fossils by University of Pennsylvania researchers has revealed that a number of specimens of the genus Psittacosaurus — once believed to represent three different species — are all members of a single species. The differences among the fossil remains that led other scientists to label them as separate species in fact arose from how the animals were buried and compressed, the study found.

"Because of the vagaries of fossilization, no two fossils are the same," said senior author Peter Dodson, professor of anatomy in Penn's School of Veterinary Medicine and professor of paleontology in the School of Arts and Sciences' Department of Earth and Environmental Science. "Animals are alive and they die, but what's crucial in paleontology is what happens to the animals after they die."

The research involved a cutting-edge technique, known as three-dimensional geometric morphometrics, which uses lasers to generate data about the shape of different specimens. This is the first time the approach has been used to study dinosaur fossils and could lead to a re-examination of the taxonomic classifications of additional dinosaur species as well as other long-extinct fossil organisms.

Brandon Hedrick, a doctoral student in the Department of Earth and Environmental Science, led the study in collaboration with Dodson. Their research will be reported in the journal PLOS ONE.

The investigation focused on dinosaurs in the genus Psittacosaurus, a word that comes from the Greek for "parrot lizard." The group was named for the animal's beaked face, not unlike that of a turtle. Originally discovered in 1923, 15 species have been classified as Psittacosaurus, though a recent analysis confirmed only nine of these as definite members of the genus. These animals were small plant-eaters that lived 120 to 125 million years ago. Paleontologists have discovered Psittacosaurus fossils in Mongolia, China and Russia and possibly in Thailand.

"Meat-eaters are sexy; plant-eaters are not," Dodson said. "This isn't a flashy dinosaur. But it has an interesting feature in that it's one of the most abundant dinosaurs known to science."

Indeed, many hundreds of Psittacosaurus specimens have been found. This abundance made the genus ideal for Hedrick and Dodson's comparative study, as it is easier to determine relationships within and between species when there are more individuals to compare.

"For example, if you have a single dachshund and a single beagle, they may appear to be different species until you found 40 dachshund-beagle mixes of various grades to examine," Hedrick said.

The scientists examined Psittacosaurus skulls discovered in the fossilized ashes of the Lujiatun beds of northeastern China's Yixian Formation. Paleontologists had previously identified the skulls as belonging to three different species, Psittacosaurus lujiatunensis, P. major or Hongshanosaurus houi.

To compare and contrast the specimens, the researchers used two techniques. First they conducted a traditional study in which they examined every skull that had been classified as one of those three species — a total of 74 specimens — for a variety of characteristics that had been used in prior studies to distinguish the species. The Penn team also compared these fossils to skulls that had been classified as belonging to eight other Psittacosaurus species.

Next they completed a more high-tech analysis of 30 skulls from the three named species. Using a hand-held stylus that captures a point in space relative to a transmitter, they pinpointed 56 "landmarks," or particular anatomical locations, on each fossil and compared the relative position of those marks between specimens. They also used a hand-held, laser-emitting scanner to make a three-dimensional image of each specimen, similar to a CT scan, from which they also collected landmark data.

Based on the "old-fashioned" method of examining the physical skulls, the researchers concluded that the three purported species were in fact one. They propose that all three can be considered members of the species P. lujiatunensis.

Results from the geometric morphometric analysis, though not sufficient on its own to classify species, supported this conclusion and suggested that how an animal's body was crushed as it fossilized — from the top, from the side or twisted — could lead to inaccurate species determinations.

"Our study found all of these false 'species' that are not biological species but are apparent species caused by the process of fossilization," Dodson said.

The Penn team said their investigation shows the value of traditional taxonomic analysis, while also revealing the potential of a new approach to analyzing fossils.

"Hopefully this will open up the paleontological community to using three-dimensional geometrics morphometrics in a variety of ways," Hedrick said. "This technique has limitless applications to understanding dinosaurs."

Article Source : University of Pennsylvania
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Green sea turtles ingesting more plastic

Sydney - Endangered green turtles are ingesting more man-made debris, including potentially lethal plastic products, than ever before, a new Australian study has shown.

The majestic turtles are significantly more likely to swallow plastic than they were in the 1980s, the study, published in the journal Conservation Biology, showed.

The research reviewed scientific literature on the ingestion of man-made rubbish in the ocean by sea turtles published since 1985.

Endangered

It showed that six of the world's seven species of sea turtles have been found to ingest debris, and all six are listed as globally vulnerable or endangered.

"We found that for green sea turtles, the likelihood that a sea turtle has ingested debris has nearly doubled in the last 25 years," Qamar Schuyler from the University of Queensland, who led the study, told AFP on Friday.

"Specifically for green turtles, it does appear that they are eating a lot more debris than they used to."

The study found that the likelihood of a green turtle, which can grow to 1.5 metres and live for 80 years, ingesting debris jumped from about 30% in 1985 to nearly 50% in 2012.

The research said it was clear that since the first data was recorded more than 100 years ago, the amount of refuse leatherback turtles had ingested had also increased.

However, between 1985 and 2012 their intake had been stable.

Plastic products eaten by turtles and other marine life can be lethal, killing the animals by either blocking their stomachs and starving them or through puncturing their intestinal system.

Schuyler said ingested plastics could also be releasing toxins into the animals, either via chemicals in the plastics themselves or which the products have absorbed as they have floated around the ocean.

"The animal may not die of that right away but it may impact things like their reproductive cycle and that has longer-term consequences," she said.

Global response

Schuyler, a doctoral candidate, said the data showed that turtles washing up with lots of plastic in them were not necessarily found in the most polluted or populated places.

"So it means that they are ingesting that debris usually somewhere farther away from where they end up," she said, adding that this suggested that a global response was needed to counter the problem.

"What we really need to look at is a large scale movement to stop debris entering the oceans."

The research, analysing 37 studies published from 1985 to 2012 which reported on data collected from before 1900 through to 2011, found that turtles in nearly all regions ingested debris, most commonly plastic.

"Our results show clearly that debris ingestion by sea turtles is a global phenomenon of increasing magnitude," the study said.

- AFP

Source :

Scientists Visualize How Cancer Chromosome Abnormalities Form in Living Cells

In new research, scientists have directly observed events that lead to formation of a chromosome abnormality that is often found in cancer cells. (Screenshot from a YouTube video, available at:http://www.youtube.com/watch?v=rPS49ZaeLag) (Credit: Image courtesy of National Cancer Institute (NCI) at NIH)

For the first time, scientists have directly observed events that lead to the formation of a chromosome abnormality that is often found in cancer cells. The abnormality, called a translocation, occurs when part of a chromosome breaks off and becomes attached to another chromosome.  The results of this study, conducted by scientists at the National Cancer Institute (NCI), part of the National Institutes of Health, appeared Aug. 9, 2013, in the journal Science. 

Chromosomes are thread-like structures inside cells that carry genes and function in heredity. Human chromosomes each contain a single piece of DNA, with the genes arranged in a linear fashion along its length.

For the first time, scientists have directly observed events that lead to formation of a chromosome abnormality that is often found in cancer cells. The results of this study--from senior author Tom Mistelli, Ph.D., lead author Vassilis Roukos, Ph.D., and colleagues at the National Cancer Institute--appears in the Aug 9, 2013 issue of the journal Science

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Translocations are very rare events, and the scientists’ ability to visualize their occurrence in real time was made possible by recently available technology at NCI that enables investigators to observe changes in thousands of cells over long time periods. “Our ability to see this fundamental process in cancer formation was possible only because of access to revolutionary imaging technology,” said the study’s senior author, Tom Misteli, Ph.D., Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, NCI.Chromosome translocations have been found in almost all cancer cells, and it has long been known that translocations can play a role in cancer development. However, despite many years of research, just exactly how translocations form in a cell has remained a mystery. To better understand this process, the researchers created an experimental system in which they induced, in a controlled fashion, breaks in the DNA of different chromosomes in living cells. Using sophisticated imaging technology, they were then able to watch as the broken ends of the chromosomes were reattached correctly or incorrectly inside the cells.

The scientists involved with this study were able to demonstrate that translocations can occur within hours of DNA breaks and that their formation is independent of when the breaks happen during the cell division cycle. Cells have built-in repair mechanisms that can fix most DNA breaks, but translocations occasionally occur.

To explore the role of DNA repair in translocation formation, the researchers inhibited key components of the DNA damage response machinery within cells and monitored the effects on the repair of DNA breaks and translocation formation. They found that inhibition of one component of DNA damage response machinery, a protein called DNAPK-kinase, increased the occurrence of translocations almost 10-fold. The scientists also determined that translocations formed preferentially between pre-positioned genes.

“These observations have allowed us to formulate a time and space framework for elucidating the mechanisms involved in the formation of chromosome translocations,” said Vassilis Roukos, Ph.D., NCI, and lead scientist of the study. 

“We can now finally begin to really probe how these fundamental features of cancer cells form,” Misteli added.

This research was supported by the Intramural Research Program of the NCI’s Center for Cancer Research.

Article Source : National Cancer Institute (NCI) at NIH.
By Science and Universe ( Nandan)