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Scooped by Dr. Stefan Gruenwald!

Engineered ribosomes could pave way for designer enzymes and synthetic cells

Engineered ribosomes could pave way for designer enzymes and synthetic cells | Amazing Science |

By hijacking the cellular machinery that makes proteins, bioengineers have developed a tool that could allow them to better understand protein synthesis, explore how antibiotics work and convert cells into custom chemical factories.

All life owes its existence to the ribosome, a huge, hardworking molecular machine that reads RNA templates transcribed from DNA, and uses the information to string together amino acids into proteins. A cell requires functioning ribosomes to survive — but they are difficult to engineer. If the engineered molecules deviate too far from the standard design, the cell will die.

“An engineered ribosome learns to do better what you want, but it starts to forget how to do its normal job,” says biochemist Alexander Mankin of the University of Illinois in Chicago.

Mankin teamed up with biochemical engineer Michael Jewett of Northwestern University in Evanston, Illinois, and others to create a ribosome that engineers could tinker with. The results of their handiwork are published in Nature1.

Ribosomes are conglomerates of RNA and protein, hundreds of times larger than typical enzymes. RNA is thought to be responsible for the bulk of a ribosome’s work, which is is considerable — it produces protein at a rate of up to 20 amino acids a second with a remarkably low error rate. “The ribosome deserves all possible respect,” says Mankin.

It is these properties that draw the attention of bioengineers such as Jewett. These researchers would like to create ribosomes that could do other chemical reactions and spit out novel polymers, or incorporate unnatural amino acids into proteins that could be used as drugs.

Each ribosome contains two clumps of snarled RNA molecules, a small subunit and a large one. The subunits come together to translate a messenger RNA sequence into protein, and then separate. They assemble again when it is time to make another protein, although not necessarily with the same partners. “In a way they are very promiscuous,” says Mankin.

That promiscuity hindered efforts to engineer ribosomes to incorporate unnatural amino acids or other compounds. Engineered and natural subunits mixed and matched, reducing the cell's ability to produce normal proteins. The solution, Mankin and Jewett's team decided, was to marry together two engineered subunits. It was unclear whether the approach would work: it was thought that ribosomes exist in two distinct units because it is necessary for their function.

The researchers used a strand of RNA to tether the large and the small subunit together, toiling for months to get the length and location of the link just right so that the machine could still function. “We certainly came close, several times, to saying ‘OK, biology wins',” says Jewett. The team screened its tethered ribosomes in Escherichia coli cells that lacked functioning RNA, and eventually found engineered ribosomes that worked well enough to support some growth, albeit slow. They then tested their platform to confirm that a tethered ribosome could operate side-by-side with natural ribosomes.

The result unlocks a molecular playground for bioengineers: by tethering the artificial subunits together, they can tweak the engineered machines to their liking without halting cell growth, says Joseph Puglisi, a structural biologist at Stanford University in California. Puglisi hopes to harness the system to study how the ribosome functions. James Collins, a bioengineer at the Massachusetts Institute of Technology in Cambridge, says that his lab may use the system to study antibiotics — many of which work by binding to bacterial ribosomes.

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Next-Gen Shotgun Cloning Using the Gibson Assembly Method

Next-Gen Shotgun Cloning Using the Gibson Assembly Method | Amazing Science |
Traditional cloning and sequencing methods can be laborious, expensive, and time-consuming techniques, especially when applied to large sample numbers. Even for the routine cloning of small sample sizes, however, many research laboratories have yet to discover the power, ease, and efficiency of the Gibson Assembly® method. First described by Dan Gibson at the J. Craig Venter Institute (JCVI) in 2009, the Gibson Assembly method is a sequence-independent, seamless cloning method that offers many advantages over traditional cloning, most notably the ability to assemble multiple DNA fragments quickly, accurately, and efficiently in a single-tube reaction.

  • SGI-DNA, a Synthetic Genomics company, offers Gibson Assembly reagent kits: the Gibson Assembly HiFi 1-Step Kit can be used for the simultaneous assembly of up to 5 fragments and the Gibson Assembly Ultra Kit can be used for the simultaneous assembly of up to 15 fragments. The Gibson Assembly method can be leveraged for a variety of applications, including routine cloning, site-directed mutagenesis, and whole-genome synthesis.
  • Here, we discuss the results and advantages of coupling the Gibson Assembly method with shotgun cloning and next-generation sequencing. The combined technologies of the Gibson Assembly method, shotgun cloning, and next-generation sequencing constitute Gibson Assembly next-generation shotgun cloning.
  • Shotgun cloning and next-generation sequencing methods offer significant time and cost savings relative to traditional (Sanger) sequencing methods and independent, repetitive cloning methods. Shotgun cloning drastically reduces sample number and next-generation sequencing drastically reduces the amount of time required for high-throughput sample processing. Combining the highly-efficient, error-correcting Gibson Assembly method with shotgun cloning and next-generation sequencing harnesses the advantages of the combined technologies, yielding substantial time and resource savings in comparison to traditional methods.
Traditional cloning and sequencing methods require the processing of individual samples through the following steps:

Preparation of insert and vector DNA by PCR amplification or restriction enzyme digestion → Vector Dephosphorylation → Ligation Cloning → Transformation → Plating → Picking Colonies → Plasmid Preparation  → Sequencing → Analysis → Construct Retrieval

The sequencing and cloning of n constructs (where n = the number of constructs) requires individually manipulating all n samples through the 10 workflow steps outlined on the previous page in n tubes (i.e., cloning 12 constructs requires handling 12 samples during every workflow stage). See Figure 1A for a schematic overview.

Combining Gibson Assembly shotgun cloning with next-generation sequencing is achieved by processing samples through the following steps:

Preparation of insert and vector DNA by PCR amplification → Gel Purification → The Gibson Assembly Method → Transformation → Plating → Picking Colonies → Plasmid Preparation → Next-Generation Sequencing → Analysis → Construct Retrieval

In the Gibson Assembly next-generation shotgun cloning workflow, samples are pooled prior to gel purification and processed in size-correlated batches. Therefore, to sequence and clone n samples using Gibson Assembly shotgun cloning, n constructs will be individually PCR-amplified. Following amplification, however, samples are pooled. For convenience and processing using 96-well plates, pools are typically batched with 8 samples per batch. Because of batching, for the remaining workflow steps, instead of processing n samples, only n/8 sample batches are manipulated for each step, which translates into substantial reagent savings (see Figures 1B & 2).
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Simple technology makes CRISPR-Cas9 gene editing cheaper

Simple technology makes CRISPR-Cas9 gene editing cheaper | Amazing Science |

University of California, Berkeley, researchers have discovered a much cheaper and easier way to target a hot new gene editing tool, CRISPR-Cas9, to cut or label DNA. The CRISPR-Cas9 technique, invented three years ago at UC Berkeley, has taken genomics by storm, with its ability to latch on to a very specific sequence of DNA and cut it, inactivating genes with ease. This has great promise for targeted gene therapy to cure genetic diseases, and for discovering the causes of disease.

The technology can also be tweaked to latch on without cutting, labeling DNA with a fluorescent probe that allows researchers to locate and track a gene among thousands in the nucleus of a living, dividing cell. The newly developed technique now makes it easier to create the RNA guides that allow CRISPR-Cas9 to target DNA so precisely. In fact, for less than $100 in supplies, anyone can make tens of thousands of such precisely guided probes covering an organism’s entire genome. The process, which they refer to as CRISPR-EATING – for “Everything Available Turned Into New Guides” – is reported in a paper to appear in the August 10 issue of the journal Developmental Cell.

As proof of principle, the researchers turned the entire genome of the common gut bacterium E. coli into a library of 40,000 RNA guides that covered 88 percent of the bacterial genome. Each RNA guide is a segment of 20 RNA base pairs: the template used by CRISPR-Cas9 as it seeks out complementary DNA to bind and cut.

These libraries can be employed in traditional CRISPR-Cas9 editing to target any specific DNA sequence in the genome and cut it, which is what researchers do to pin down the function of a gene: knock it out and see what bad things happen in the cell. This can help pinpoint the cause of a disease, for example. The process is called genetic screening and is done in batches: each of the thousands of probes is introduced into a single cell on a plate filled with hundreds of thousands of cells.

“We can make these libraries for a lot less money, which makes genetic screening potentially accessible in organisms less well studied,” such as those that have not yet had their genomes sequenced, said first author Andrew Lane, a UC Berkeley post-doctoral fellow.

But Lane and colleague Rebecca Heald, UC Berkeley professor of molecular and cell biology, developed the technology in order to track chromosomes in real-time in living cells, in particular during cell division, a process known as mitosis. This is part of a larger project by Heald to find out what regulates the size of the nucleus and other subcellular components as organisms grow from just a few cells to many cells.

“This technology will allow us to paint a whole chromosome and look at it live and really follow it in the nucleus during the cell cycle or as it goes through developmental transitions, for example in an embryo, to see how it changes in size and structure,” Heald said.

The new technique uses standard PCR (polymerase chain reaction) to generate many short lengths of DNA from whatever segment of DNA a researcher is interested in, up to and including an entire genome.

These fragments are then precisely snipped at a region called a PAM, which is critical to CRISPR binding. Simple restriction enzymes are then used to cut each piece 20 base pairs from the PAM end, generating the exact size of RNA guide that CRISPR uses in searching the genome for complementary sites. These guide RNAs are then easily incorporated into the CRISPR-Cas9 complex, yielding tens of thousands of probes for labeling or cutting DNA.

“By using the genome itself as a source for guide RNAs, their approach puts the creation of libraries that target contiguous regions in reach of almost any lab,” said Jacob Corn, managing and scientific director of the Innovative Genomics Initiative at UC Berkeley. “This could be very useful for genome imaging and certain kinds of screens, and I’m very interested to see how it enables biological discovery using Cas9 tools.”

Sophia Nguyen's curator insight, July 28, 11:01 PM

As before, CRISPR is something that I am interested in studying since it is relatively new in the gene editing world. Also, UC Berkeley is a college that I am considering  applying and attending so it would be good to contribute to this research.

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Virginia Tech scientist develops model for robots with bacterial brains

Virginia Tech scientist develops model for robots with bacterial brains | Amazing Science |

Forget the Vulcan mind-meld of the Star Trek generation — as far as mind control techniques go, bacteria is the next frontier.

In a paper published today in Scientific Reports, which is part of the Nature Publishing Group, a Virginia Tech scientist used a mathematical model to demonstrate that bacteria can control the behavior of an inanimate device like a robot. “Basically we were trying to find out from the mathematical model if we could build a living microbiome on a nonliving host and control the host through the microbiome,” said Warren Ruder, an assistant professor of biological systems engineering in both the College of Agriculture and Life Sciences and the College of Engineering

"We found that robots may indeed be able to function with a bacterial brain,” he said. For future experiments, Ruder is building real-world robots that will have the ability to read bacterial gene expression levels in E. coli using miniature fluorescent microscopes. The robots will respond to bacteria he will engineer in his lab.

On a broad scale, understanding the biochemical sensing between organisms could have far reaching implications in ecology, biology, and robotics. In agriculture, bacteria-robot model systems could enable robust studies that explore the interactions between soil bacteria and livestock. In healthcare, further understanding of bacteria’s role in controlling gut physiology could lead to bacteria-based prescriptions to treat mental and physical illnesses. Ruder also envisions droids that could execute tasks such as deploying bacteria to remediate oil spills.

The findings also add to the ever-growing body of research about bacteria in the human body that are thought to regulate health and mood, and especially the theory that bacteria also affect behavior.

The study was inspired by real-world experiments where the mating behavior of fruit flies was manipulated using bacteria, as well as mice that exhibited signs of lower stress when implanted with probiotics.

Ruder’s approach revealed unique decision-making behavior by a bacteria-robot system by coupling and computationally simulating widely accepted equations that describe three distinct elements: engineered gene circuits in E. coli, microfluid bioreactors, and robot movement.

The bacteria in the mathematical experiment exhibited their genetic circuitry by either turning green or red, according to what they ate. In the mathematical model, the theoretical robot was equipped with sensors and a miniature microscope to measure the color of bacteria telling it where and how fast to go depending upon the pigment and intensity of color.

The model also revealed higher order functions in a surprising way. In one instance, as the bacteria were directing the robot toward more food, the robot paused before quickly making its final approach — a classic predatory behavior of higher order animals that stalk prey.

Ruder’s modeling study also demonstrates that these sorts of biosynthetic experiments could be done in the future with a minimal amount of funds, opening up the field to a much larger pool of researchers.

Via Integrated DNA Technologies
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Zebrafish reveal drugs that may improve bone marrow transplant

Zebrafish reveal drugs that may improve bone marrow transplant | Amazing Science |

sing large-scale zebrafish drug-screening models, Harvard Stem Cell Institute (HSCI) researchers at Boston Children’s Hospital have identified a potent group of chemicals that helps bone marrow transplants engraft or “take.” The findings, featured on the cover of the today’s issue of Nature, could lead to human trials in patients with cancer and blood disorders within a year or two, says senior investigator Leonard Zon, a member of the HSCI executive committee and a professor in Harvard’s Department of Stem Cell and Regenerative Biology.

The compounds, known as epoxyeicosatrienoic acids, or EETs, boosted stem cell engraftment in both zebrafish and mice and could make human bone marrow transplants more efficient. Better engraftment could also allow umbilical cord blood to be used as an alternative to marrow as a source of blood stem cells, greatly increasing a patient’s chances of finding a matched donor and enhancing safety.

“Ninety percent of cord blood units can’t be used because they’re too small,” explains Zon, who directs the Stem Cell Program at Boston Children’s. “If you add these chemicals, you might be able to use more units. Being able to get engraftment allows you to pick a smaller cord blood sample that might be a better match.”

EETs are fats that appear to work by stimulating cell migration. They were among the top hits in a screen of 500 known compounds conducted in Boston Children’s newly upgraded Karp Aquatics Facility. While zebrafish have previously led Zon’s team to compounds that boost blood stem cell numbers, such as prostaglandin (currently in several clinical trials under the name ProHema), the new drug screen specifically tested the stem cells’ transplantability and engraftment.

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Fluorescent Proteins and the Creation of a Living Laser Inside Living Cells

Fluorescent Proteins and the Creation of a Living Laser Inside Living Cells | Amazing Science |

A few years back, a pair of researchers at Massachusetts General Hospital made human cells glow by impregnating them with a molecule that's normally found in jellyfish called green fluorescent protein (GFP) and packing them into a resonant cavity that amplified the amount of light each cell produced. Now, according to a new study recently published in the journal Nano Letters, a team of scientists from the University of St Andrews have developed a means of making individual glowing cells also act as their own resonant cavities.

The St. Andrews team accomplished this by coaxing each cell to engulf a tiny plastic bubble (the green dot in the image above) that acts as a resonant cavity. Each bubble is precisely sized and imbued with fluorescent dye. When a laser hits the cell, it excites the dye which bounces around and amplifies inside the bubble, then fluoresces at a different wavelength. Interestingly, the color of the light that the cell emits depends on the size of the bubble. So far, the researchers have gotten cells to produce light at three different wavelengths. And while the team has only been able to get the method to work in petri dishes, they hope to further develop it into a means of tracking specific cells -- say, tumor cells -- for days, even weeks.

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New Tool for Investigating RNA: Sticky-flare nanotechnology exposes RNA misregulation in living cells

New Tool for Investigating RNA: Sticky-flare nanotechnology exposes RNA misregulation in living cells | Amazing Science |

RNA is a fundamental ingredient in all known forms of life -- so when RNA goes awry, a lot can go wrong. RNA misregulation plays a critical role in the development of many disorders, such as mental disability, autism and cancer.

A new technology -- called “Sticky-flares” -- developed by nanomedicine experts at Northwestern University offers the first real-time method to track and observe the dynamics of RNA distribution as it is transported inside living cells.

Sticky-flares have the potential to help scientists understand the complexities of RNA better than any analytical technique to date and observe and study the biological and medical significance of RNA misregulation. Details will be published this week in the journal Proceedings of the National Academy of Sciences (PNAS).

Previous technologies made it possible to attain static snapshots of RNA location, but that isn’t enough to understand the complexities of RNA transport and localization within a cell. Instead of analyzing snapshots of RNA to try to understand functioning, Sticky-flares help create an experience that is more like watching live-streaming video.

“This is very exciting because much of the RNA in cells has very specific quantities and localization, and both are critical to the cell’s function, but until this development it has been very difficult, and often impossible, to probe both attributes of RNA in a live cell,” said Chad A. Mirkin, a nanomedicine expert and corresponding author of the study. “We hope that many more researchers will be able to use this platform to increase our understanding of RNA function inside cells.”

Mirkin is the George B. Rathmann Professor of Chemistry in the Weinberg College of Arts and Sciences and professor of medicine, chemical and biological engineering, biomedical engineering and materials science and engineering.

Sticky-flares are tiny spherical nucleic acid gold nanoparticle conjugates that can enter living cells and target and transfer a fluorescent reporter or “tracking device” to RNA transcripts. This fluorescent labeling can be tracked via fluorescence microscopy as it is transported throughout the cell, including the nucleus.

In the PNAS paper, the scientists explain how they used Sticky-flares to quantify β–actin mRNA in HeLa cells (the oldest and most commonly used human cell line) as well as to follow the real-time transport of β–actin mRNA in mouse embryonic fibroblasts.

Sticky-flares are built upon another technology from Mirkin’s group called NanoFlares, which was the first genetic-based approach that is able to detect live circulating tumor cells out of the complex matrix that is human blood.

NanoFlares have been very useful for researchers that operate in the arena of quantifying gene expression. AuraSense, Inc., a biotechnology company that licensed the NanoFlare technology from Northwestern University, and EMD-Millipore, another biotech company, have commercialized NanoFlares. There are now more than 1,700 commercial forms of NanoFlares sold under the SmartFlare™ name in more than 230 countries.

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Shape-shifting molecular cousins are the key to DNA repair

Shape-shifting molecular cousins are the key to DNA repair | Amazing Science |

It’s taken years of frustration and dedication (not to mention countless hours spent in a small room roughly the temperature of a domestic fridge) but the hard work has finally paid off. Our researchers Martin Taylor and Simon Boulton at the Francis Crick Institute have solved a decades-old biological mystery, publishing the results of their molecular sleuthing in the journal Cell.

The story centres on a protein called RAD51 and its closely-related molecular ‘cousins’, known as paralogs, which are involved in helping cells repair DNA damage. It’s been known for some time that people who inherit a single faulty version of one of the genes that makes these proteins are at higher risk of developing breast and ovarian tumors – for example, our researchers found that inheriting a mistake in a paralog gene called RAD51D increases a woman’s chances of developing ovarian cancer six-fold. Inheriting two broken copies causes a severe genetic syndrome known as Fanconi anaemia, which usually leads to leukaemia. But until now, exactly how RAD51’s molecular cousins work – and how they contribute to cancer – was completely unknown, until no.

Via Integrated DNA Technologies
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Researchers develop basic computing elements for bacteria

Researchers develop basic computing elements for bacteria | Amazing Science |
Sensors, memory switches, and circuits can be encoded in a common gut bacterium.

The “friendly” bacteria inside our digestive systems are being given an upgrade, which may one day allow them to be programmed to detect and ultimately treat diseases such as colon cancer and immune disorders.

In a paper published today in the journal Cell Systems, researchers at MIT unveil a series of sensors, memory switches, and circuits that can be encoded in the common human gut bacterium Bacteroides thetaiotaomicron.

These basic computing elements will allow the bacteria to sense, memorize, and respond to signals in the gut, with future applications that might include the early detection and treatment of inflammatory bowel disease or colon cancer.

Researchers have previously built genetic circuits inside model organisms such as E. coli. However, such strains are only found at low levels within the human gut, according to Timothy Lu, an associate professor of biological engineering and of electrical engineering and computer science, who led the research alongside Christopher Voigt, a professor of biological engineering at MIT.

“We wanted to work with strains like B. thetaiotaomicron that are present in many people in abundant levels, and can stably colonize the gut for long periods of time,” Lu says. The team developed a series of genetic parts that can be used to precisely program gene expression within the bacteria. “Using these parts, we built four sensors that can be encoded in the bacterium’s DNA that respond to a signal to switch genes on and off inside B. thetaiotaomicron,” Voigt says. These can be food additives, including sugars, which allow the bacteria to be controlled by the food that is eaten by the host, Voigt adds.

To sense and report on pathologies in the gut, including signs of bleeding or inflammation, the bacteria will need to remember this information and report it externally. To enable them to do this, the researchers equipped B. thetaiotaomicron with a form of genetic memory. They used a class of proteins known as recombinases, which can record information into bacterial DNA by recognizing specific DNA addresses and inverting their direction.

The researchers also implemented a technology known as CRISPR interference, which can be used to control which genes are turned on or off in the bacterium. The researchers used it to modulate the ability of B. thetaiotaomicron to consume a specific nutrient and to resist being killed by an antimicrobial molecule.

The researchers demonstrated that their set of genetic tools and switches functioned within B. thetaiotaomicron colonizing the gut of mice. When the mice were fed food containing the right ingredients, they showed that the bacteria could remember what the mice ate.

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New Treatment for Melanoma Uses a Form of the Herpes Virus

New Treatment for Melanoma Uses a Form of the Herpes Virus | Amazing Science |

The American Cancer Society estimates that about 74,000 Americans will be diagnosed with melanoma this year and almost 10,000 will die from this deadliest form of skin cancer. Over the past several years, treatment of advanced cases of melanoma has been transformed as new FDA-approved therapies developed by several different companies have come onto the market. An FDA advisory committee recently approved a therapy that takes a totally novel approach that involves injecting a live attenuated virus directly into regionally or distant metastatic melanoma tumors.

HSV-1 infections cause cold sores and sometimes genital herpes, although infection with human simplex virus 2 is more often the cause of genital herpes. Researchers have characterized the virulence genes of the virus. Talimogene laherparepvec, sometimes shortened to T-VEC, is made by depleting those virulence genes and inserting sequences that generate GM-CSF. It’s believed that removal of the virulence genes decreases the chances that the virus will infect nerve cells and will instead home in on tumor cells. By delivering GM-CSF, the genetically engineered virus enhances tumor antigen presentation to the immune system and induction of immune system attack on the malignancy.

Encouraging durable response results

Talimogene laherparepvec was studied in a randomized, open label phase 3 study to compare the new therapy with GM-CSF injections in subjects with unresectable stage IIIB, IIIC, and IV melanoma. A total of 437 subjects were randomized into the study at 64 study sites. The study was designed to demonstrate an improvement in durable response rate, which was defined as a complete response or partial response maintained for at least six months. Subjects were to receive therapy until Week 24, even if their melanoma was progressing. GM-CSF was used for comparison purposes because at the time that this study was designed, it was also in clinical studies as a treatment for melanoma. It is unclear, though, if GM-CSF by itself has any therapeutic value.

To be enrolled in the study, people had to be age 18 or older, have a histologically confirmed malignant melanoma of the stages listed in the previous paragraph, measurable disease of at least 1 cm, injectable disease (either on the surface of the skin or through the use of ultrasound guidance), ECOG performance of 0 or 1, and a life expectancy greater than four months from date of randomization. The study exclusions included active cerebral disease, any bone metastases, history of secondary cancer unless disease-free for at least five years, open herpetic skin lesions, and primary ocular or mucosal melanoma.

Via Krishan Maggon
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Expanding the DNA alphabet: ‘extra’ DNA base (5-formylcytosine) found to be stable in mammals

Expanding the DNA alphabet: ‘extra’ DNA base (5-formylcytosine) found to be stable in mammals | Amazing Science |
A rare DNA base, previously thought to be a temporary modification, has been shown to be stable in mammalian DNA, suggesting that it plays a key role in cellular function.

Researchers from the University of Cambridge and the Babraham Institute have found that a naturally occurring modified DNA base appears to be stably incorporated in the DNA of many mammalian tissues, possibly representing an expansion of the functional DNA alphabet.

The new study, published in the journal Nature Chemical Biology, has found that this rare ‘extra’ base, known as 5-formylcytosine (5fC) is stable in living mouse tissues. While its exact function is yet to be determined, 5fC’s physical position in the genome makes it likely that it plays a key role in gene activity.

“This modification to DNA is found in very specific positions in the genome – the places which regulate genes,” said the paper’s lead author Dr Martin Bachman, who conducted the research while at Cambridge’s Department of Chemistry. “In addition, it’s been found in every tissue in the body – albeit in very low levels.”

“If 5fC is present in the DNA of all tissues, it is probably there for a reason,” said Professor Shankar Balasubramanian of the Department of Chemistry and the Cancer Research UK Cambridge Institute, who led the research. “It had been thought this modification was solely a short-lived intermediate, but the fact that we’ve demonstrated it can be stable in living tissue shows that it could regulate gene expression and potentially signal other events in cells.”

Since the structure of DNA was discovered more than 60 years ago, it’s been known that there are four DNA bases: G, C, A and T (Guanine, Cytosine, Adenine and Thymine). The way these bases are ordered determines the makeup of the genome. In addition to G, C, A and T, there are also small chemical modifications, or epigenetic marks, which affect how the DNA sequence is interpreted and control how certain genes are switched on or off. The study of these marks and how they affect gene activity is known as epigenetics.

5fC is one of these marks, and is formed when enzymes called TET enzymes add oxygen to methylated DNA – a DNA molecule with smaller molecules of methyl attached to the cytosine base. First discovered in 2011, it had been thought that 5fC was a ‘transitional’ state of the cytosine base which was then being removed from DNA by dedicated repair enzymes. However, this new research has found that 5fC can actually be stable in living tissue, making it likely that it plays a key role in the genome.

Using high-resolution mass spectrometry, the researchers examined levels of 5fC in living adult and embryonic mouse tissues, as well as in mouse embryonic stem cells – the body’s master cells which can become almost any cell type in the body.

They found that 5fC is present in all tissues, but is very rare, making it difficult to detect. Even in the brain, where it is most common, 5fC is only present at around 10 parts per million or less. In other tissues throughout the body, it is present at between one and five parts per million.

The researchers applied a method consisting of feeding cells and living mice with an amino acid called L-methionine, enriched for naturally occurring stable isotopes of carbon and hydrogen, and measuring the uptake of these isotopes to 5fC in DNA. The lack of uptake in the non-dividing adult brain tissue pointed to the fact that 5fC can be a stable modification: if it was a transient molecule, this uptake of isotopes would be high.

The researchers believe that 5fC might alter the way DNA is recognised by proteins. “Unmodified DNA interacts with a specific set of proteins, and the presence of 5fC could change these interactions either directly or indirectly by changing the shape of the DNA duplex,” said Bachman. “A different shape means that a DNA molecule could then attract different proteins and transcription factors, which could in turn change the way that genes are expressed.”

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Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA

Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA | Amazing Science |

Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development. However, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, a group of scientists reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. The researchers observed the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. The presented method allows researchers to control the pore size between nanowires by varying the number of nanowire growth cycles and to select the pore size of the nanowires based on the analytical range of the target biomolecules.

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Transplantability of a circadian clock to a noncircadian organism

Transplantability of a circadian clock to a noncircadian organism | Amazing Science |

Circadian oscillators are post-translationally regulated and affect gene expression in autotrophic cyanobacteria. Oscillations are controlled by phosphorylation of the KaiC protein, which is modulated by the KaiA and KaiB proteins. However, it remains unclear how time information is transmitted to transcriptional output. A group of researchers now show reconstruction of the KaiABC oscillator in the noncircadian bacterium Escherichia coli. This orthogonal system shows circadian oscillations in KaiC phosphorylation and in a synthetic transcriptional reporter. Coexpression of KaiABC with additional native cyanobacterial components demonstrates a minimally sufficient set of proteins for transcriptional output from a native cyanobacterial promoter in E. coli. Together, these results demonstrate that a circadian oscillator is transplantable to a heterologous organism for reductive study as well as wide-ranging applications.

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Researchers build bacteria's photosynthetic engine

Researchers build bacteria's photosynthetic engine | Amazing Science |

Nearly all life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Oxygen-producing plants and cyanobacteria perfected this process 2.7 billion years ago. But the first photosynthetic organisms were likely single-celled purple bacteria that began absorbing near-infrared light and converting it to sulfur or sulfates about 3.4 billion years ago.

Found in the bottom of lakes and ponds today, purple bacteria possess simpler photosynthetic organelles—specialized cellular subunits called chromatophores—than plants and algae. For that reason, Klaus Schulten of the University of Illinois at Urbana–Champaign (UIUC) targeted the chromatophore to study photosynthesis at the atomic level.

As a computational biophysicist, Schulten unites biologists' experimental data with the physical laws that govern the behavior of matter. This combination allows him to simulate biomolecules, atom by atom, using supercomputers. The simulations reveal interactions between molecules that are impossible to observe in the laboratory, providing plausible explanations for how molecules carry out biological functions in nature.

In 2014, a team led by Schulten used the Titan supercomputer, located at the US Department of Energy's (DOE's) Oak Ridge National Laboratory, to construct and simulate a single chromatophore. The soccer ball-shaped chromatophore contained more than 100 million atoms—a significantly larger biomolecular system than any previously modeled. The project's scale required Titan, the flagship supercomputer at the Oak Ridge Leadership Computing Facility (OLCF), a DOE Office of Science User Facility, to calculate the interaction of millions of atoms in a feasible time frame that would allow for data analysis.

"For years, scientists have seen that cells are made of these machines, but they could only look at part of the machine. It's like looking at a car engine and saying, 'Oh, there's an interesting cable, an interesting screw, an interesting cylinder.' You look at the parts and describe them with love and care, but you don't understand how the engine actually works that way," Schulten said. "Titan gave us the fantastic level of computing we needed to see the whole picture. For the first time, we could go from looking at the cable, the screw, the cylinder to looking at the whole engine."

Schulten's chromatophore simulation is being used to understand the fundamental process of photosynthesis, basic research that could one day lead to better solar energy technology. Of particular interest: how hundreds of proteins work together to capture light energy at an estimated 90 percent efficiency.

Via LeapMind, Jocelyn Stoller
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Host-Targeted Antibodies Effective Against Malaria

Host-Targeted Antibodies Effective Against Malaria | Amazing Science |

All antimalarial drugs produced to date target the disease-causing parasite, but a new study in the Journal of Experimental Medicine shows that drugs which target host proteins are also a potential avenue for new interventions.

This study targets a protein that the most deadly malaria parasite, Plasmodium falciparum, relies on to invade human red blood cells. Targeting this human protein blocks an essential interaction, and can wipe out an established malaria infection in mice in less than three days.

Targeting host factors may help researchers overcome one of the biggest challenges to malaria control: drug resistance. Drug resistance arises due to genetic changes in the rapidly-evolving Plasmodium falciparum parasite, which, in Southeast Asia, has rendered one of the current front-line antimalarials, artemisinin, largely ineffective. Researchers are battling to find a solution before the resistant strains spread to other malaria endemic areas, including Africa, a region that accounts for 90 per cent of malaria deaths worldwide. By targeting host factors, rather than the parasite factors, the researchers believe that parasites are far less likely to develop resistance to the new drug.

"This counter-intuitive approach to malaria treatment leaves the parasite powerless," explains Dr. Zenon Zenonos, a first author from the Wellcome Trust Sanger Institute. "If the parasite can't bind to the surface of our red blood cells and invade, it can't reach the next stage in its lifecycle, so it dies. There's nothing the parasite can do to get round it, as the interaction is absolutely essential for infection to occur."

PfRH5, a protein required by the malaria parasite, needs to bind to basigin, a protein that is displayed on the outer surface of human red blood cells, for the cell to become infected. Blockade of the PfRH5-basigin interaction renders the parasite unable to enter red blood cells, and therefore the infection is wiped out.

"When we discovered the PfRH5-basigin interaction in 2011, we knew we had found a chink in the malaria parasite's armour, the question was how to exploit it," says Dr Gavin Wright, corresponding author from the Wellcome Trust Sanger Institute. "Using PfRH5 in a vaccine is one approach, but we were also interested to see if we could disrupt the interaction in the opposite direction rather than by conventionally targeting the parasite. This has significant advantages in preventing the ability of the parasite to develop resistance."

To study the likely human response to therapy, the antibody targeting basigin described in this study was tested in humanised mice that have had the majority of their immune cells and blood cells replaced with those from their human counterparts. In the mice, levels of infection fell to essentially undetectable levels within 72 hours of being treated with low doses of the antibody targeting basigin. Importantly, no side toxic effects were observed in the mouse models that were treated with the antibody in these experiments.

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Startup Company Says It’s the First to Make Synthetic Spider Silk

Startup Company Says It’s the First to Make Synthetic Spider Silk | Amazing Science |

For years, material scientists have been trying to figure out a way to give consumers broad access to the benefits of spider silk. As a naturally occurring supermaterial, spider silk is five times stronger than steel and more elastic than rubber bands, which suggests some amazing potential use cases, including bulletproof vests, biodegradable water bottles, and flexible bridge suspension ropes. But so far, every group that’s attempted to produce enough of the stuff to bring it to the mass market, from researchers to giant corporations, has pretty much failed.

The problem is there’s no way to get the silk from spiders themselves—creatures known to be territorial and cannibalistic, which doesn’t lend itself to raising them in groups. So people have had to resort to creative workarounds. They’ve tried raising genetically engineered silkworms, or inserting genes into microorganisms to express the needed spider silk protein. All of these efforts, however, have seen little success. Spider silk protein is complex, and even when experimenters are able to create fibers, these come out so fine that entirely new spinning systems need to be invented from scratch to turn the strands into thread. It doesn’t keep groups from trying though, and every few months or so, it seems, news of some spider silk breakthrough goes viral, only to quiet down after a few months. And consumers keep waiting.

But today, after five years of quiet operation, a startup called Bolt Threads has emerged to claim it’s made meaningful progress on the challenge. The Emeryville, California-based company grew out of the graduate school studies of three scientists from the University of California, San Francisco and UC Berkeley, and it has raised $40 million so far from such notable investors as Foundation Capital, Formation 8 and Founders Fund, as well as from government grants from institutions like the National Science Foundation. If its founders are to be believed, Bolt Threads may have solved the mystery—finally—of how to make spider silk commercially plausible.

“Basically, our mission from the beginning was to make a scalable amount of spider silk and bring that to consumers,” CEO Dan Widmaier tells WIRED. “It’s a problem that’s been around for a long time, and has been hampered entirely by technical challenges.” Widmaier knows it’s a bold claim. That’s why, he says, the company chose to fly under the radar for so long. “We decided to keep our heads down and try to solve the problem before we went out and started talking about all the cool things we can do with the technology,” he says. “Now, we’re ready to say we’re here.”

Widmaier says that generally speaking, what they do isn’t new in the world of biotechnology. The scientists genetically engineered a microorganism that can yield large quantities of silk protein through a yeast fermentation process—not just grams of silk protein, but metric tons. Then, using a proprietary mechanical system, a wet silk protein solution is manually squeezed through small extrusion holes and goes into a liquid bath that turns the stuff into solid fibers. While Widmaier won’t give away the minute specifications of how it all works, he does say that the extrusion process mimics the behavior of a spider’s spinneret—its silk-spinning organ. The naturally occurring spinning process has been the other key problem would-be spider silk producers have had difficulty mimicking in the past.

The result, Widmaier claims, is a technology that can artificially recreate the remarkably strong protein fibers spiders make. On top of that, he says, the fibers can even be tuned to possess different properties on demand: the researchers simply change the protein sequence on the platform to tweak the qualities of the material according to preference. Widmaier says they can make spider silk that’s stronger, stretchier, or waterproof, for example, depending on preference. “What we’ve learned is we could prod nature a little bit in the lab and engineer these new properties in,” says Widmaier.

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Novel therapeutic strategy for single gene disorders delivers RNA that encodes the missing protein

Novel therapeutic strategy for single gene disorders delivers RNA that encodes the missing protein | Amazing Science |

Researchers have demonstrated the feasibility of delivering an RNA that encodes for the protein alpha-1-antitrypsin (AAT)--which is missing or nonfunctional in the genetic disorder AAT deficiency--into cells in the laboratory, enabling the cells to produce highly functional AAT. This innovative approach to treating single gene disorders such as AAT deficiency offers and safe, simpler, and more cost-effective alternative to gene therapy or protein replacement, according to the authors of the study published in Nucleic Acid Therapeutics, a peer-reviewed journal from Mary Ann Liebert, Inc., publishers. The article is available free on the Nucleic Acid Therapeutics website until August 27, 2015.

In the article "In vitro Evaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency", Tatjana Michel, Stefanie Krajewski, and coauthors, University Medical Center, Tuebingen, Germany, produced a messenger RNA sequence that cells can translate to generate the AAT protein. The researchers assessed the stability and utility of the encapsulated RNA over time and evaluated the amount of AAT protein produced by the cells and how well the protein functioned. The data show no negative effects of the transfected RNA on the viability of the cells and no immune activation.

"The field is looking for advances in modified mRNA as a therapeutic strategy," says Executive Editor Graham C. Parker, PhD, The Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine, Children's Hospital of Michigan, Detroit, MI. "Demonstrations such as this from the University Medical Center, Tuebingen, Germany, show real progress."

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Are 'Stem Cell Factories' The Future Of Regenerative Medicine? Scientists Eye Possible Breakthrough

Are 'Stem Cell Factories' The Future Of Regenerative Medicine? Scientists Eye Possible Breakthrough | Amazing Science |

Scientists have found a synthetic substrate that has ability to produce billions of stem cells, a new study showed. The findings could potentially pave way for creation of "stem cell factories," which can be used in treatment of the heart, liver, and brain.

Researchers at the University of Nottingham in the U.K. developed the cost-effective substrate that allows the growth of stem cells, and can also survive long-term storage.  Findings of the study were published in the June issue of Advanced Materials journal.

Chris Denning, study co-author and a professor of stem cell biology at the University of Nottingham, explained that a person loses about 5 billion cells during a heart attack and in order to replace those cells, doctors need about 10 to 15 billion stem cells as some of the cells do not survive or differentiate into heart cells. 

Researchers also said that patients with eye disorders in some countries have already receive stem cell-derived treatments.

"The field of regenerative medicine has snowballed in the last five years and over the coming five years a lot more patients will be receiving stem cell treatments," Denning said. "Clinical trials are still in the very early stages. However, with this kind of product, if we can get it commercialized and validated by the regulators, it could be helping patients in two to three years."

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Researchers folded DNA into the shape of a nanoscale bunny and other 3D structures

Researchers folded DNA into the shape of a nanoscale bunny and other 3D structures | Amazing Science |

Folding DNA into the shape of a tiny bunny rabbit is now easier than ever, according to a study published in Nature today. Folding DNA isn’t new — it’s known as DNA origami — but automating the process is. Thanks to a set of computer algorithms, researchers have developed a way to streamline the design phase that comes before the DNA assembly — a substantial step toward 3D printing at the nanoscale.

This has not been done before, it is novel and surprising," says Thorsten Schmidt, a chemist at the Dresden University of Technology who didn't work on the study. "In fact, we have a very related study under review at the moment and the only bad aspect of Björn Högberg’s study is that they were faster than us."

The bunny, while cute, wasn’t the point of the study. Rather, it’s a demonstration that scientists can automatically generate a DNA sequence to form a complex shape — the closest thing to 3D printing on a very tiny scale. "It’s almost a one-click procedure," Högberg says. And if scientists can fully automate the process, they’ll have a real DNA printer at their disposal — one that could, among other things, make drugs easier to deliver to the right places in the body.

Actually, there are a lot of ideas about how these techniques could be used. In addition to drug delivery, researchers are working on coating the DNA structures with non-biological materials, like gold, that react when the structure comes in contact with light.

But at this point, the bunny and the bottle don't do all that much. "We're not really concerned with the genetic information," Högberg says. "We're using DNA purely as a construction material."

Now that the study has been published, the researchers want to find a way to make their own construction materials. That may mean using natural DNA — taken from a plant or bacteria that they cultivate themselves — instead of synthetic DNA, Högberg says. "We're getting very good at making structures at the nanoscale," Högberg says. Researchers just need to find a way to make lots tiny DNA bunnies cheaply — and all at once.

Via Integrated DNA Technologies
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DNA Origami: Novel rare structures built from DNA emerge

DNA Origami: Novel rare structures built from DNA emerge | Amazing Science |

Scientists have worked for many years to refine the technique of DNA origami. The aim is to compose new sets of design rules, vastly expanding the range of nanoscale architectures generated by the method. In a new research, a variety of innovative nanoforms are described, each displaying unprecedented design control. The technique promises to bring futuristic microelectronics and biomedical innovations to market.

Hao Yan, a researcher at Arizona State University's Biodesign Institute, has worked for many years to refine the technique. His aim is to compose new sets of design rules, vastly expanding the range of nanoscale architectures generated by the method. In new research, a variety of innovative nanoforms are described, each displaying unprecedented design control.

In the current study, complex nano-forms displaying arbitrary wireframe architectures have been created, using a new set of design rules. "Earlier design methods used strategies including parallel arrangement of DNA helices to approximate arbitrary shapes, but precise fine-tuning of DNA wireframe architectures that connect vertices in 3D space has required a new approach," Yan says.

Yan has long been fascinated with Nature's seemingly boundless capacity for design innovation. The new study describes wireframe structures of high complexity and programmability, fabricated through the precise control of branching and curvature, using novel organizational principles for the designs. (Wireframes are skeletal three-dimensional models represented purely through lines and vertices.)

The resulting nanoforms include symmetrical lattice arrays, quasicrystalline structures, curvilinear arrays, and a simple wire art sketch in the 100-nm scale, as well as 3D objects including a snub cube with 60 edges and 24 vertices and a reconfigurable Archimedean solid that can be controlled to make the unfolding and refolding transitions between 3D and 2D.

In previous investigations, the Yan group created subtle architectural forms at an astonishingly minute scale, some measuring only tens of nanometers across--roughly the diameter of a virus particle. These nano-objects include spheres, spirals, flasks, Möbius forms, and even an autonomous spider-like robot capable of following a prepared DNA track.

Although DNA origami originally produced nanoarchitectures of purely aesthetic interest, refinements of the technique have opened the door to a range of exciting applications including molecular cages for the encapsulation of molecules, enzyme immobilization and catalysis, chemical and biological sensing tools, drug delivery mechanisms, and molecular computing devices.

The technique described in the new study takes this approach a step further, allowing researchers to overcome local symmetry restrictions, creating wireframe architectures with higher order arbitrariness and complexity. Here, each line segment and vertex is individually designed and controlled. The number of arms emanating from each vertex may be varied from 2 to 10 and the precise angles between adjacent arms can be modified.

In the current study, the method was first applied to symmetrical, regularly repeating polygonal designs, including hexagonal, square and triangular tiling geometries. Such common designs are known as tessellation patterns. A clever strategy involving a series of bridges and loops was used to properly route the scaffold strand, allowing it to pass through the entire structure, touching all lines of the wireframe once and only once. Staple strands were then applied to complete the designs.

In subsequent stages, the researchers created more complex wireframe structures, without the local translational symmetry found in the tessellation patters. Three such patterns were made, including a star shape, a 5-fold Penrose tile and an 8-fold quasicrystalline pattern. (Quasicrystals are structures that are highly ordered but non-periodic. Such patterns can continuously fill available space, but are not translationally symmetric.) Loop structures inserted into staple strands and unpaired nucleotides at the vertex points of the scaffold strands were also used, allowing researchers to perform precision modifications to the angles of junction arms.

The new design rules were next tested with the assembly of increasingly complex nanostructures, involving vertices ranging from 2 to 10 arms, with many different angles and curvatures involved, including a complex pattern of birds and flowers. The accuracy of the design was subsequently confirmed by AFM imaging, proving that the method could successfully yield highly sophisticated wireframe DNA nanostructures.

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Revolutionizing the already revolutionary technology of optogenetics

Revolutionizing the already revolutionary technology of optogenetics | Amazing Science |
In most optogenetics experiments, light is delivered to cells from lasers through fiber optic cables. A new research project focuses on making cells produce their own light, letting them control their own activity or the activity of neighboring cells.

First introduced in a practical form in 2005, optogenetics gave brain scientists the amazing new capability to use pulses of laser light to control almost any type of neuron in any area with precise timing. Prior means of controlling neurons weren't ideal. Electrical pulses were powerful but drove all the cells in an area, not just desired cell types. Drugs couldn't confine control to a particular area and didn't have precise timing. Optogenetics could do it all by genetically engineering cells to become excited or suppressed by different colors of light.

But optogenetics can still do more, said Christopher Moore, associate professor of neuroscience at Brown, who leads the new project, funded by a new $1-million grant from the W.M. Keck Foundation. His goal is to make cells "smart" enough to emit light precisely when needed in order to optogenetically control themselves or their neighbors. If optogenetics is ever approved for human use, this new form of self-regulation—which would not involve injecting light into the body from outside—could produce new ways to treat problems ranging from epileptic seizures to Parkinson's disease to diabetes.

In 2013, Moore's collaborator Ute Hochgeschwender, associate professor at CMU, demonstrated how to make optogenetic cells emit their own light using a capability widely found in nature: bioluminescence.

Bioluminescence is the natural chemical reaction that allows fireflies and many sea creatures to make light. The advance of pairing bioluminescence with optogenetics allowed scientists to make the technology wireless. In most optogenetic experiments, laser light is delivered into the body by a fiber optic cable, but with BL-OG—shorthand for BioLuminescent-OptoGenetics—a cool, biologically compatible light could be triggered in cells just by administering a drug. Now the team of Moore, Hochgeschwender, and Brown professors Barry Connors, Julie Kauer and Diane Lipscombe, interim director of the Brown Institute for Brain Science, will pursue the next big step.

The team plans to try out that idea by focusing on the flow of calcium ions among cells in various parts of the body, a particular area of Lipscombe's expertise. Calcium excites cells, such as neurons, by building up positive charges in them until they cross a threshold for action. In epilepsy, for example, there is too much of that excitation. Connors has studied that in detail.

Here is how the team proposes to help: With BL-OG, they already know how to make target cells capable of emitting and/or responding to light. The next step is to link those capabilities to sensing the levels of calcium ions. Synthetic biology, in which scientists add snippets of DNA instructions to the genome of a cell or whole organism to essentially program in a new capability, offers one potential way to make that feasible.

In the example of epilepsy, BL-OG-enabled neurons in the brain could be programmed to glow red (like a traffic light) if calcium ions are surging in too quickly. That red glow could trigger neighboring optogenetic cells to dampen their excitation amid the calcium buildup, effectively stopping a seizure as soon as it starts.

"A similar effect could normalize brain activity in Parkinson's disease, where runaway bursting in specific brain areas is thought to underlie the symptoms of that disorder," Moore said. Moreover, optogenetics works in other parts of the body. In the pancreas, the team hopes to see if it is possible to program BL-OG-capable cells to sense low blood glucose levels. Calcium ions have an important role in insulin secretion, so when sugars are too low, cells programmed to be self-regulating could trigger a glow of light to optogenetically increase the excitation of cells involved in signaling insulin production.

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The bioprinted ‘play dough’ capable of cell and protein transfer

The bioprinted ‘play dough’ capable of cell and protein transfer | Amazing Science |
Scientists have developed a new technique allowing the bioprinting at ambient temperatures of a strong paste similar to ‘play dough’ capable of incorporating protein-releasing microspheres.

The scientists demonstrated that the bioprinted material, in the form of a micro-particle paste capable of being injected via a syringe, could sustain stresses and strains similar to cancellous bone – the ‘spongy’ bone tissue typically found at the end of long bones.

This work, published today (3 July 2015) in the journal Biofabrication, suggests that bioprinting at ambient temperatures is a viable route to the production of materials for bone repair which would allow the inclusion of cells and proteins capable of accelerating the healing of large fractures.

“Bioprinting is a hot research area in tissue engineering,” explains Dr Jing Yang, of the University of Nottingham, a lead author on the paper. “However it usually requires a printing environment that isn’t compatible with living cells – and those materials that are compatible with living cells usually don’t have sufficient mechanical properties for certain applications.”

“Initially we’re targeting the clinical application of this material as injectable bone defect filler,” continues Dr Yang, “but we’ve postulated that its properties would make it highly suitable for use as a scaffold to reconstruct larger shapes, which could help with more complicated reconstructions – such as nasal reconstruction.”

Typically, bioprinting techniques involve high temperature processes, or the application of ultraviolet light or organic solvents, all of which prevent the incorporation of cells and therapeutic biomolecules during the fabrication process.

This technique involved blending poly(L-lactic-co-glycolic acid) and polyethylene glycol with carrier fluids at room temperature to form a micro-particulate extrudable paste that can be formed to desired shapes. These pastes were incubated at 37 °C to form porous solid constructs. The next steps of the process will be to apply this process in a clinical application.
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Why Crispr-Cas9 Gene-Editing Technology Has Scientists Excited

Why Crispr-Cas9 Gene-Editing Technology Has Scientists Excited | Amazing Science |
Researchers are exploring the idea of treating disease by replacing the defective gene causing the trouble

A new technology for “editing” defective genes has raised hopes for a future generation of medicines treating intractable diseases like cancer, cystic fibrosis and sickle-cell anemia. Such drugs could home in on a specific gene causing a disease, then snip it out and, if necessary, replace it with a healthy segment of DNA. Drugs of this type wouldn’t hit the mass market for years, if ever; pharmaceutical firms are only now exploring how to make drugs using the gene-editing technology, called Crispr-Cas9. But the approach offers tremendous potential for developing new treatments for diseases caused by a mutated gene.

“What if you could go right to the root cause of that disease and repair the broken gene? That’s what people are excited about,” says Katrine Bosley, chief executive of privately held Editas Medicine. Its projects include developing a gene-editing drug treating one type of Leber congenital amaurosis, a rare disease that causes blindness in children.

Crispr-Cas9 isn’t the only technology capable of editing genes, but researchers consider it easier to use than other methods, says Dana Carroll, a professor of biochemistry at the University of Utah School of Medicine, who helped pioneer another gene-editing approach called zinc finger nucleases.

Among other efforts under way using Crispr-Cas9 technology, privately held Intellia Therapeutics Inc., in partnership with Novartis AG, is probing how to create a gene-editing drug that could harness the immune system to fight certain blood cancers. The two companies are also exploring the treatment of hereditary blood disorders like sickle-cell anemia and beta thalassemia.

Intellia CEO Nessan Bermingham says drugs based on Crispr-Cas9 promise to complement the pills and biotech drugs currently available, targeting diseases that aren’t well treated by existing therapies. “This is a new tool to target and treat disease,” he says. Industry and academic laboratories are also using the technology for more immediate effect: to genetically engineer mice and other animals so that they have humanlike diseases that researchers can then readily study.

Using Crispr-Cas9 to make the animal models is “much quicker, easier than the other methods that have been available,” says Tim Harris, senior vice president of precision medicine at Biogen Inc. The company is using the technology to study amyotrophic lateral sclerosis, or Lou Gehrig’s disease, which has lacked good animal models.

Crispr-Cas9 attracted notoriety in April, when Chinese scientists reported trying to repair the genes that cause beta thalassemia in 86 human embryos obtained from a fertilization clinic. The work raised fears that gene editing could be used to tweak babies in many ways before they were born.

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New Candidate Brings us One Step Closer to an HIV Vaccine

New Candidate Brings us One Step Closer to an HIV Vaccine | Amazing Science |

Despite more than 30 years of intense research, a cure or vaccine for HIV still continues to elude us. But scientists are not quitting, and slowly but surely they seem to be making promising progress in this field. For example, two new mouse studies have just come out that demonstrate that a novel vaccine candidate is able to prompt the beginnings of an immune reaction needed to prevent infection. While the results are not the “breakthrough” everyone is looking for, they are certainly a stride in the right direction.

Vaccines can be made in a variety of different ways, for example by inactivating whole pathogens or isolating particular components of them, both with the ultimate goal of stimulating a defense response from the immune system, readying it for any future assaults. But the problem with pesky HIV is that it mutates remarkably rapidly, changing its components so that they become unrecognizable by the immune system. This means that should a vaccine be successful in inducing the production of protective antibodies, they usually have such a narrow window of activity that they are effectively useless.

But there are some antibodies that are different, called broadly neutralizing antibodies (bNAbs), and scientists have high hopes that these may hold the key to producing a successful HIV vaccine. As the name suggests, rather than being specific to just one target, these antibodies are able to recognize and inhibit a range of HIV variants, or strains, and thus are much more therapeutically useful. Although a subset of HIV-positive individuals produce these antibodies, scientists have so far failed to induce their production via vaccination.

Many researchers believe the key to achieving this is by presenting the body with multiple targets, or antigens, that differ slightly, training the immune system to recognize and hone in on the more conserved elements of HIV that are found in different strains. One particular molecule that scientists are interested in is an antigen called eOD-GT8, which was engineered by researchers, headed by William Schief, at The Scripps Research Institute.

Rather than attempting to directly elicit bNAbs, this antigen is designed to stimulate the production of precursor antibodies that will eventually mature into bNAbs following prolonged exposure to the virus, The Scientist explains. So by starting off with these immature antibodies, scientists hypothesize it may be possible to encourage them to develop into bNAbs over time by gradually exposing the immune system to slightly different HIV antigens, forcing the antibodies to mutate in order to recognize more conserved regions of the virus.

When testing this molecule out in mice genetically engineered to produce antibodies similar to those found in humans, the researchers found that it was indeed able to elicit these first-line antibodies. Additionally, they found it also created a pool of antibody-producing “memory” B cells that the researchers believe could be boosted through exposure to different antigens, sort of like receiving booster shots. These findings have been reported in Science.

Via Steven Krohn
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First working synthetic immune organ with controllable antibodies

First working synthetic immune organ with controllable antibodies | Amazing Science |

Cornell University engineers have created a functional, synthetic immune organoid (a lab-grown ball of cells with some of the features of a normal organ) that produces antibodies. The engineered organ has implications for everything from rapid production of immune therapies to new frontiers in cancer or infectious disease research. The first-of-its-kind immune organoid was created in the lab of Ankur Singh, assistant professor of mechanical and aerospace engineering, who applies engineering principles to the study and manipulation of the human immune system.

The synthetic organ is bio-inspired by secondary immune organs like the lymph node or spleen. It is made from a hydrogel (a soft, nanocomposite gelatin-like biomaterial), reinforced with silicate nanoparticles to keep the structure from melting at body temperature. This biomaterial is also seeded with B cells. It mimics the body’s normal anatomical microenvironment of lymphoid tissue, which produces lymphocytes and antibodies in the lymph nodes, thymus, tonsils, and spleen.

Like a real organ, the organoid converts B cells — which make antibodies that respond to infectious invaders — into germinal centers, which are clusters of B cells that activate, mature and mutate their antibody genes when the body is under attack. Germinal centers are a sign of infection and are not present in healthy immune organs.

The engineers have demonstrated how they can control this immune response in the organ and tune how quickly the B cells proliferate, get activated and change their antibody types. According to their paper, their 3-D organ outperforms existing 2-D lab cultures and can produce activated B cells up to 100 times faster.

According to Singh, the organoid could lead to increased understanding of B cell functions, an area of study that typically relies on animal models to observe how the cells develop and mature, and could also be used to study specific infections and how the body produces antibodies to fight those infections — from Ebola to HIV. “You can use our system to force the production of immunotherapeutics at much faster rates,” he said. Such a system also could be used to test toxic chemicals and environmental factors that contribute to infections or organ malfunctions.

The process of B cells becoming germinal centers is not well understood, and in fact, when the body makes mistakes in the genetic rearrangement related to this process, blood cancer can result. “In the long run, we anticipate that the ability to drive immune reaction ex vivo [outside the body] at controllable rates grants us the ability to reproduce immunological events with tunable parameters for better mechanistic understanding of B cell development and generation of B cell tumors, as well as screening and translation of new classes of drugs,” Singh said.

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