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Posts Tagged ‘genetic engineering’


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Leigh syndrome is one of the hundreds of so-called mitochondrial diseases, which are caused by defects in the mitochondria that produce 90 percent of the body’s energy. These disorders are rare; about 1,000 to 4,000 babies in the United States are born with one every year. But they are devastating and can result in grave impairment of nearly any bodily system. They are largely untreatable, uniformly incurable and very difficult to screen.

 

Leigh syndrome is a terrible disease. It emerges shortly after birth and claims one major organ after another. Movement becomes difficult, and then impossible. A tracheotomy and feeding tube are often necessary by toddlerhood, and as the disease progresses, lungs frequently have to be suctioned manually. Most children with the condition die by the age of 5 or 6.

 

Scientists have devised a procedure called mitochondrial replacement therapy (M.R.T.) that involves transplanting the nucleus of an affected egg (mitochondrial diseases are passed down from the mother’s side) into an unaffected one whose nucleus has been removed. The procedure is sometimes called “three-parent in vitro fertilization”. Mitochondria contain a minuscule amount of DNA, any resulting embryo would have mitochondrial DNA from the donor egg and nuclear DNA from each of its parents.

 

After decades of careful study in cell and animal research M.R.T. is now finally being tested in human clinical trials by doctors in Britain (no births confirmed yet officially). In the United States, however, this procedure is effectively illegal. M.R.T. does not involve altering any genetic code. Defective mitochondria are swapped out for healthy ones.

 

Mitochondrial DNA governs only a handful of basic cellular functions. It is separate from nuclear DNA, which helps determine individual traits like physical appearance, intelligence and personality. That means M.R.T. cannot be used to produce the genetically enhanced “designer babies” and thus should be allowed in humans. But, there is no way to know how safe or effective M.R.T. is until doctors and scientists test it in humans.

 

References:

 

 

https://pharmaceuticalintelligence.com/2016/10/07/the-three-parent-technique-to-avoid-mitochondrial-disease-in-embryo/

 

 

 

 

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Stem Cells Used as Delivery Truck for Brain Cancer Drugs

Reporter: Irina Robu, PhD

Medulloblastoma, common brain cancer in children has been very difficult to treat therapeutically with traditional interventions which relies on surgical techniques to remove the bulk of the cancerous tissue. The researchers seen the need for novel treatments of medulloblastomas that have recurred, as well as for treatments that are less toxic overall. For this reason, data from University of North Carolina (UNC) Lineberger Comprehensive Cancer Center and  Eshelman School of Pharmacy published a study in PLOS named “Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection”, validates how cancer-hunting stem cells can track down and deliver a drug to terminate medulloblastoma cells hiding after surgery.

The technology in the research is an extension of a discovery that won researchers a Nobel Prize in 2012 and showed they could transform skin cells into stem cells. The research team started by reprogramming skin cells into stem cells and genetically engineered them to manufacture a substance that becomes toxic to other cells when exposed to another drug. Inserting the drug carries the stem cells into the brain of laboratory models after surgery decreased the size of tumors by 15 times and extended median survival in mice by 133%.

In this study, the scientists indicated they could shrink tumors in murine models of medulloblastoma, hence extending the rodents life. The approach holds promise for reducing side effects and helping more children with medulloblastoma. Amazingly the researchers also developed a laboratory model of medulloblastoma that allowed them to simulate the way standard care is currently delivered—surgery followed by drug therapy. Using this model, they discovered that after surgically removing a tumor, the cancer cells that remained grew faster.

According to the study investigator, Shawn Hingtgen, PhD, the cells are like a FedEx truck that will deliver cytotoxic agents directly into the tumor to a particular location. In earlier studies, Dr. Hingtgen and his colleagues showed that they could flip skin cells into stem cells that hunt and transport cancer-killing drugs to glioblastoma, the deadliest malignant brain tumor in adults.

Medulloblastoma is cancer that happens mostly in kids between ages of three and eight, and while current therapy has changed survival pretty dramatically, it can still be pretty toxic. The ability to use a patient’s own cells to target the tumor directly would be “the holy grail” of therapy, the investigators trust it could hold capacity for other rare, and sometimes fatal, brain cancer types that occur in children as well.

Source

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198596

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Intestinal inflammatory pharmaceutics

Curator: Larry H. Bernstein, MD, FCAP

 

AbbVie Invests in Synthetic Microbes for Treatment of Intestinal Disorders

Aaron Krol    http://www.bio-itworld.com/2016/2/10/abbvie-invests-synthetic-microbes-treatment-intestinal-disorders.html

 

February 10, 2016 | This morning, AbbVie announced a partnership with Synlogic of Cambridge, Mass., to create microbiome-based therapies for the treatment of inflammatory bowel disease (IBD). The two companies have sketched out a suggested three-year timeline for preclinical research and development, after which AbbVie will take over advancing any drug candidates into clinical trials.

Drugs inspired by the microbes that live in the human gut are a hot topic in biotech. Companies like Seres Health and Vedanta Biosciences are pursuing the idea from a variety of angles, from making traditional small molecule drugs that interact with the microbiome, to creating probiotics or microbial cocktails that restore a healthy balance to the gut ecosystem. IBD, including Crohn’s disease and ulcerative colitis, is an especially popular target for these companies, thanks to strong suggestions that bacterial populations can affect the course of the disease. Already, Second Genome and Coronado Biosciences have taken prospective treatments into the clinic (though the latter has been dealt serious setbacks in Phase II trials).

But even among this peculiar batch of startups, Synlogic’s approach to drug design is exquisitely odd. The company calls its products “synthetic biotics”―in fact, they’re genetically engineered bacteria whose DNA contains intricately designed “gene circuits,” built to start producing therapeutic molecules when and only when the patient needs them.

“We are not looking at correcting the dysregulation of microbes in the gut, like other microbiome companies,” CEO José-Carlos Gutiérrez-Ramos tells Bio-IT World. “We have one bacterium, and it’s engineered to do different functions.”

Synlogic was founded in 2013 by two synthetic biologists at MIT, Timothy Lu and Jim Collins. (Bio-IT World has previously spoken with Lu about his academic work on bacterial gene circuits.) Gutiérrez-Ramos joined almost two years later, leaving a position as the head of Pfizer’s BioTherapeutics R&D group, where he had plenty of opportunity to turn emerging biotechnology ideas into drug candidates ready for submission to the FDA.

Still, synthetic biotics are a good deal more unusual than the biologic drugs he worked on at Pfizer.

His new company doesn’t quite spin functions for its microbes out of whole cloth. All the genes the company uses are copied either from the human genome, or from the bacteria living inside us. But by recombining those genes into circuits, Gutiérrez-Ramos believes Synlogic can finely control whether and when genes are expressed, giving its synthetic biotics the same dosage control as a traditional drug. Meanwhile, choosing the right bacterium to engineer―the current favorite is a strain called E. coli Nissle―ensures the biotics do not form stable colonies in the gut, but can be cleared out as soon as a patient stops treatment.

“We’re pharma guys,” he says. “What we want is to have pharmacologically well-defined products.”

The Molecular Circuit Board

Even before the partnership with AbbVie, Synlogic had a pipeline of drug candidates in development, all meant to treat rare genetic disorders caused by single mutations that shut down the activity of a crucial gene. In principle, there seems to be no reason that bacteria carrying the right genes couldn’t pick up the slack. “We know the patient is missing a function that is typically performed by the liver, or the kidney, or the pancreas,” says Gutiérrez-Ramos. “What we do is shift that function from an organ to a stable fraction of the microbiome.”

The approach is in some ways analogous to gene therapy, where a corrected version of a broken gene is inserted into a patient’s own DNA. “We don’t use that word, but the fact is it’s a non-somatic gene therapy,” Gutiérrez-Ramos says. “And if something goes wrong, you can control it just by stopping treatment.” The most advanced synthetic biotic in Synlogic’s pipeline targets urea cycle disorder, exactly the sort of disease that might otherwise be addressed by gene therapy: patients are missing a single enzyme that helps remove nitrogen from the body and prevent it from forming ammonia in the bloodstream. Synlogic will meet with the FDA this March to discuss whether and how this first product can be tested in humans.

Gutierrez Ramos

The new IBD program with AbbVie, however, adds a whole new level of complexity. Executives from the two companies have been in discussions for around six months, and both agree that no single mechanism will be enough to provide significant relief for patients. Crohn’s and ulcerative colitis are painful autoimmune diseases that involve both a weakening of the epithelial lining in the stomach, and a buildup of inflammatory molecules. The development plan that AbbVie and Synlogic have agreed on includes three separate methods of attack to relieve these symptoms.

“One approach AbbVie is very interested in is for our synthetic biotics to produce substances that could tighten the epithelial barrier,” says Gutiérrez-Ramos. “Another approach is to degrade pro-inflammatory molecules”―the same tack taken by AbbVie’s current leading IBD drug, Humira, which targets the inflammatory protein TNFα. “Finally, we can produce anti-inflammatory molecules.”

Uniquely, synthetic biotics can perform all three functions at once; it’s just a matter of inserting the right genes. But that alone might not be a decisive advantage over some sort of combination therapy. The biggest selling point of Synlogic’s microbes is not the genes they can be engineered to express―what you might call the “output” of their gene circuits―but the input, the DNA elements called “inducible promoters” that decide when those genes should be activated.

The core idea is that patients will have a constant population of synthetic biotics in their bodies, taken daily―but those microbes will only generate their therapeutic payloads when needed. In IBD, Gutiérrez-Ramos explains, “it’s not that the patient is always inflamed, but they have flares. Our vision, and AbbVie’s vision, is that the bacteria that you take every day sense when the flare is coming, and then trigger the genetic output.”

This would be a major improvement over a drug like Humira, which after all is constantly inhibiting a part of the immune system. Patients taking Humira, or one of the many other immunosuppressant drugs for IBD, are at a constantly heightened risk of infection; tuberculosis is a particular specter for these patients. If Synlogic can find a genetic “on-switch” that responds to a reliable indicator of IBD flares, it could potentially create a much more precisely administered treatment, while still giving patients the simple dosing schedule of one pill every day.

The company has leads on two inducible promoters that might do the trick: one that reacts to nitric oxide, and another tied to reactive oxygen species. Of course, there’s no guarantee that either will respond sensitively to IBD flares in a real clinical setting. “This is an early time for the technology,” says Gutiérrez-Ramos. “We have demonstrated this in animals, but we have to demonstrate it in humans.”

Although it’s far too early to say if synthetic biotics will become an ordinary part of the pharma toolkit, AbbVie’s decision to invest in the technology offers the means to test this approach on a large scale. Synlogic expects to raise its own funding for trials of its rare disease products, which the FDA does not expect to enroll huge numbers of patients, but IBD is a problem of a very different order.

“We are very honored to work with truly the leader in treatment of inflammatory bowel disease,” says Gutiérrez-Ramos. With the backing of big pharma, it will be possible to trial microbiome-based therapies for the kinds of common, chronic diseases that are the biggest drain on our healthcare system. What’s more, the AbbVie partnership is an important signal of the industry’s faith in synthetic biology as an approach to treating disease.

 

 

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Regulatory DNA engineered

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New Type of CRISPR Screen Probes the Regulatory Genome

Aaron Krol    http://www.bio-itworld.com/2016/2/8/new-type-crispr-screen-probes-regulatory-genome.html

February 8, 2016 | When a geneticist stares down the 3 billion DNA base pairs of the human genome, searching for a clue to what’s gone awry in a single patient, it helps to narrow the field. One of the most popular places to look is the exome, the tiny fraction of our DNA―less than 2%―that actually codes for proteins. For patients with rare genetic diseases, which might be fully explained by one key mutation, many studies sequence the whole exome and leave all the noncoding DNA out. Similarly, personalized cancer tests, which can help bring to light unexpected treatment options, often sequence the tumor exome, or a smaller panel of protein-coding genes.

Unfortunately, we know that’s not the whole picture. “There are a substantial number of noncoding regions that are just as effective at turning off a gene as a mutation in the gene itself,” says Richard Sherwood, a geneticist at Brigham and Women’s Hospital in Boston. “Exome sequencing is not going to be a good proxy for what genes are working.”

Sherwood studies regulatory DNA, the vast segment of the genome that governs which genes are turned on or off in any cell at a given time. It’s a confounding area of genetics; we don’t even know how much of the genome is made up of these regulatory elements. While genes can be recognized by the presence of “start” and “stop” codons―sequences of three DNA letters that tell the cell’s molecular machinery which stretches of DNA to transcribe into RNA, and eventually into protein―there are no definite signs like this for regulatory DNA.

Instead, studies to discover new regulatory elements have been somewhat trial-and-error. If you suspect a gene’s activity might be regulated by a nearby DNA element, you can inhibit that element in a living cell, and see if your gene shuts down with it.

With these painstaking experiments, scientists can slowly work their way through potential regulatory regions―but they can’t sweep across the genome with the kind of high-throughput testing that other areas of genetics thrive on. “Previously, you couldn’t do these sorts of tests in a large form, like 4,000 of them at once,” says David Gifford, a computational biologist at MIT. “You would really need to have a more hypothesis-directed methodology.”

Recently, Gifford and Sherwood collaborated on a paper, published in Nature Biotechnology, which presents a new method for testing thousands of DNA loci for regulatory activity at once. Their assay, called MERA (multiplexed editing regulatory assay), is built on the recent technology boom in CRISPR-Cas9 gene editing, which lets scientists quickly and easily cut specific sequences of DNA out of the genome.

So far, their team, including lead author Nisha Rajagopal from Gifford’s lab, has used MERA to study the regulation of four genes involved in the development of embryonic stem cells. Already, the results have defied the accepted wisdom about regulatory DNA. Many areas of the genome flagged by MERA as important factors in gene expression do not fall into any known categories of regulatory elements, and would likely never have been tested with previous-generation methods.

“Our approach allows you to look away from the lampposts,” says Sherwood. “The more unbiased you can be, the more we’ll actually know.”

A New Kind of CRISPR Screen

In the past three years, CRISPR-Cas9 experiments have taken all areas of molecular biology by storm, and Sherwood and Gifford are far from the first to use the technology to run large numbers of tests in parallel. CRISPR screens are an excellent way to learn which genes are involved in a cellular process, like tumor growth or drug resistance. In these assays, scientists knock out entire genes, one by one, and see what happens to cells without them.

This kind of CRISPR screen, however, operates on too small a scale to study the regulatory genome. For each gene knocked out in a CRISPR screen, you have to engineer a strain of virus to deliver a “guide RNA” into the cellular genome, showing the vicelike Cas9 molecule which DNA region to cut. That works well if you know exactly where a gene lies and only need to cut it once—but in a high-throughput regulatory test, you would want to blanket vast stretches of DNA with cuts, not knowing which areas will turn out to contain regulatory elements. Creating a new virus for each of these cuts is hugely impractical.

The insight behind MERA is that, with the right preparation, most of the genetic engineering can be done in advance. Gifford and Sherwood’s team used a standard viral vector to put a “dummy” guide RNA sequence, one that wouldn’t tell Cas9 to cut anything, into an embryonic stem cell’s genome. Then they grew plenty of cells with this prebuilt CRISPR system inside, and attacked each one with a Cas9 molecule targeted to the dummy sequence, chopping out the fake guide.

Normally, the result would just be a gap in the CRISPR system where the guide once was. But along with Cas9, the researchers also exposed the cells to new, “real” guide RNA sequences. Through a DNA repair mechanism called homologous recombination, the cells dutifully patched over the gaps with new guides, whose sequences were very similar to the missing dummy code. At the end of the process, each cell had a unique guide sequence ready to make cuts at a specific DNA locus—just like in a standard CRISPR screen, but with much less hands-on engineering.

By using a large enough library of guide RNA molecules, a MERA screen can include thousands of cuts that completely tile a broad region of the genome, providing an agnostic look at anywhere regulatory elements might be hiding. “It’s a lot easier [than a typical CRISPR screen],” says Sherwood. “The day the library comes in, you just perform one PCR reaction, and the cells do the rest of the work.”

In the team’s first batch of MERA screens, they created almost 4,000 guide RNAs for each gene they studied, covering roughly 40,000 DNA bases of the “cis-regulatory region,” or the area surrounding the gene where most regulatory elements are thought to lie. It’s unclear just how large any gene’s cis-regulatory region is, but 40,000 bases is a big leap from the highly targeted assays that have come before.

“We’re now starting to do follow-up studies where we increase the number of guide RNAs,” Sherwood adds. “Eventually, what you’d like is to be able to tile an entire chromosome.”

Far From the Lampposts

Sherwood and Gifford tried to focus their assays on regions that would be rich in regulatory elements. To that end, they made sure their guide RNAs covered parts of the genome with well-known signs of regulatory activity, like histone markers and transcription factor binding sites. For many of these areas, Cas9 cuts did, in fact, shut down gene expression in the MERA screens.

But the study also targeted regions around each gene that were empty of any known regulatory features. “We tiled some other regions that we thought might serve as negative controls,” explains Gifford. “But they turned out not to be negative at all.”

The study’s most surprising finding was that several cuts to seemingly random areas of the genome caused genes to become nonfunctional. The authors named these DNA regions “unmarked regulatory elements,” or UREs. They were especially prevalent around the genes Tdgf1 and Zfp42, and in many cases, seemed to be every bit as necessary to gene activity as more predictable hits on the MERA screen.

These results caught the researchers so off guard that it was natural to wonder if MERA screens are prone to false positives. Yet follow-up experiments strongly supported the existence of UREs. Switching the guide RNAs from aTdgf1 MERA screen and a Zfp42 screen, for example, produced almost no positive results: the UREs’ regulatory effects were indeed specific to the genes near them.

In a more specific test, the researchers chose a particular URE connected to Tdgf1, and cut it out of a brand new population of cells for a closer look. “We showed that, if we deleted that region from the genome, the cells lost expression of the gene,” says Sherwood. “And then when we put it back in, the gene became expressed again. Which was good proof to us that the URE itself was responsible.”

From these results, it seems likely that follow-up MERA screens will find even more unknown stretches of regulatory DNA. Gifford and Sherwood’s experiments didn’t try to cover as much ground around their target genes as they might have, because the researchers assumed that MERA would mostly confirm what was already known. At best, they hoped MERA would rule out some suspected regulatory regions, and help show which regulatory elements have the biggest effect on gene expression.

“We tended to prioritize regions that had been known before,” Sherwood says. “Unfortunately, in the end, our datasets weren’t ideally suited to discovering these UREs.”

Getting to Basic Principles

MERA could open up huge swaths of the regulatory genome to investigation. Compared to an ordinary CRISPR screen, says Sherwood, “there’s only upside,” as MERA is cheaper, easier, and faster to run.

Still, interpreting the results is not trivial. Like other CRISPR screens, MERA makes cuts at precise points in the genome, but does not tell cells to repair those cuts in any particular way. As a result, a population of cells all carrying the same guide RNA can have a huge variety of different gaps and scars in their genomes, typically deletions in the range of 10 to 100 bases long. Gifford and Sherwood created up to 100 cells for each of their guides, and sometimes found that gene expression was affected in some but not all of them; only sequencing the genomes of their mutated cells could reveal exactly what changes had been made.

By repeating these experiments many times, and learning which mutations affect gene expression, it will eventually be possible to pin down the exact DNA bases that make up each regulatory element. Future studies might even be able to distinguish between regulatory elements with small and large effects on gene expression. In Gifford and Sherwood’s MERA screens, the target genes were altered to produce a green fluorescent protein, so the results were read in terms of whether cells gave off fluorescent light. But a more precise, though expensive, approach would be to perform RNA sequencing, to learn which cuts reduced the cell’s ability to transcribe a gene into RNA, and by how much.

A MERA screen offers a rich volume of data on the behavior of the regulatory genome. Yet, as with so much else in genetics, there are few robust principles to let scientists know where they should be focusing their efforts. Histone markers provide only a very rough sketch of regulatory elements, often proving to be red herrings on closer examination. And the existence of UREs, if confirmed by future experiments, shows that we don’t yet even know which areas of the genome to rule out in the hunt for regulatory regions.

“Every dataset we get comes closer and closer to computational principles that let us predict these regions,” says Sherwood. As more studies are conducted, patterns may emerge in the DNA sequences of regulatory elements that link UREs together, or reveal which histone markers truly point toward regulatory effects. There might also be functional clues hidden in these sequences, hinting at what is happening on a molecular level as regulatory elements turn genes on and off in the course of a cell’s development.

For now, however, the data is still rough and disorganized. For better and for worse, high-throughput tools like MERA are becoming the foundation for most discoveries in genetics—and that means there is a lot more work to do before the regulatory genome begins to come into focus.

CORRECTED 2/9/16: Originally, this story incorrectly stated that only certain cell types could be assayed with MERA for reasons related to homologous recombination. In fact, the authors see no reason MERA could not be applied to any in vitro cell line, and hope to perform screens in a wide range of cell types. The text has been edited to correct the error.

 

 

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Molecular On/Off Switches in Bacterial Design

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Controlling Synthetic Bacteria

“Kill switches” ensure that genetically engineered bacteria survive only in certain environmental conditions.

By Kate Yandell | Dec 7, 2015   http://www.the-scientist.com//?articles.view/articleNo/44715/title/Controlling-Synthetic-Bacteria/

http://www.the-scientist.com/images/News/December2015/620ecoli.jpg

FLICKR, NIAID

Two synthetic gene circuits allow researchers to keep genetically engineered (GE) microbes alive only under specific conditions, and to kill them when their services are no longer needed. The circuits, described today (December 7) in Nature Chemical Biology,could help pave the path to safe diagnostics, therapies, or environmental remediation strategies that rely on GE bacteria.

“This is yet another step forward towards better biosafety and biocontainment based on certain aspects of existing technology,” said Guy-Bart Stan, a synthetic biologist at Imperial College London who was not involved in the study.

Study coauthor James Collins, a synthetic biologist at MIT, began to design these gene circuits, or “kill switches,” after becoming interested in using GE microbes for diagnostic and therapeutic purposes. “We were motivated to begin working on the topic as synthetic biology has moved increasingly toward real-world applications,” Collins told The Scientist. Other groups are working to engineer microbes for bioremediation and industrial processes, among other things.

But with genetic modification comes the concern that scientists will create new and uncontrollable species that outcompete or share their genes with wild-type organisms, permanently altering the environment or endangering people’s health.

Earlier this year, two research teams led by Yale bioengineer Farren Isaacs and Harvard geneticist George Church showed that they could genetically modify Escherichia coli to incorporate synthetic amino acids into essential proteins. When the bacteria are not fed the amino acids, they cannot produce these essential proteins, and so they die. This strategy yields bacteria that are very unlikely to survive without support from scientists but requires intensive engineering of the bacterial genome. (See “GMO ‘Kill Switches,’” The Scientist, January 2015.)

In contrast, Collins and his colleagues set out to create kill switches that could work in a more diverse range of microbes. “Our circuit-based safeguards can be conveniently transferred to different bacterial strains without modifying the target cell’s genome,” he wrote in an email.

First, Collins and his colleagues generated a kill switch called “Deadman,” named for a locomotive braking system in which the train will only run if the engineer is affirmatively holding down a pedal. In the microbial version of Deadman, a researcher must feed bacteria a substance called anhydrotetracycline at all times, or else the microbes will express a toxin and self-destruct.

The researchers generated a genetic circuit containing genes for the proteins LacI and TetR, a toxin that is only expressed in the absence of LacI, and a protease that degrades LacI. Under normal circumstances, TetR is preferentially expressed over LacI. TetR expression also triggers expression of the protease, which degrades any LacI that has been expressed. Without LacI, cells express the toxin and die. But when the cells are fed anhydrotetracycline, TetR is inhibited and LacI is expressed. LacI represses the toxin and keeps the cells alive.

Other versions of the Deadman circuit can be designed to degrade essential proteins in the absence of anhydrotetracycline, said Collins.

A second kill switch, “Passcode,” similarly requires that researchers maintain a specific environment for cells lest they express a toxin. Passcode requires a combination of input molecules for cells to survive. The system relies on hybrid transcription factors, each with one component that recognizes a specific DNA sequence, and one component that is sensitive to specific small molecules, such as galactose or cellobiose. One hybrid transcription factor, factor C, turns off expression of a toxin. Two other hybrid transcription factors, factors A and B, suppress expression of factor C. But specific small molecules can keep them from interacting with C. Another small molecule could prevent C from repressing the toxin. Therefore, to keep the cells alive, researchers must provide them with two small molecules that keep factors A and B in check, and make sure not to give them a third small molecule that will interfere with C.

Scientists designing Passcode kill-switches could make hybrid transcription factors respond to whatever combination of small molecules they desired, said Collins. “The strength of our kill switches lies in their flexibility and their ability to detect complex environmental signals for biocontainment.” He noted that companies hoping to keep others from using their cells could keep the recipe for their feed a secret.

“The great advantage is that you can effectively scale this and create different combinations of environments that contain different cocktails of these small molecules, thereby allowing you to effectively create a suite of cells that are going to be viable in different environments,” said Isaacs.

But Church warned that Collins’s circuit-based approach might not as effectively contain bacteria as an amino acid-based method, like one his group developed, since the cells are not fundamentally dependent on foreign biology to survive.

“If you need to have the ability to really scale your containment across a number of different species, then I could see the Passcode kill switches would be incredibly valuable,” said Isaacs. “If you are very concerned about escape frequencies and your degree of biocontainment, maybe you’d opt for something where the organism has been recoded and it relies on a synthetic amino acid.”

Still, Stan said the new paper is a demonstration that creating easy-to-insert kill switches based on genetic circuits is feasible. “I think what they wanted to show in the paper is basically that using some existing genetic circuitry . . .  you can obtain biosafety for the here and now.”

 

C.T.Y. Chan et al., “‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment,” Nature Chemical Biology, doi:10.1038/nchembio.1979, 2015.  

Tags   synthetic biology, microbes, genetic engineering and biosafety

 

GMO “Kill Switches”

Scientists design bacteria reliant upon synthetic amino acids to contain genetically modified organisms.

By Kerry Grens | Jan 21, 2015   http://www.the-scientist.com/?articles.view/articleNo/41954/title/GMO–Kill-Switches-/

One of the biggest concerns about genetically modified organisms (GMOs) is that they can infiltrate wild populations and spread their altered genes among naturally occurring species. In Nature today (January 21), two groups present a new method of containing GMOs: by making some of their essential proteins reliant upon synthetic amino acids not found outside of the laboratory.

“What really makes this a valuable step change is that kill switches beforehand were very susceptible to mutation or other conditions, such as metabolic cross feeding, from basically inactivating them,” said Tom Ellis, a synthetic biologist at Imperial College London who was not involved in the studies. The new approach circumvents some of those problems by making it extremely unlikely for the genetically modified bacteria to be able to survive outside of the conditions dictated by their custom-designed genomes.

Both research teams—one led by George Church at Harvard Medical School and the other by Farren Isaacsat Yale University—based their work on so-called genetically recoded organisms (GROs), bacterial genomes that have had all instances of a particular codon replaced by another. Church and Isaacs, along with their colleagues, had previously developed this concept in collaboration. Since then, their respective groups designed the replacement codons to incorporate a synthetic amino acid, and engineered proteins essential to the organism to rely upon the artificial amino acid for proper function.

“Here, for the first time, we’re showing that we’re able to engineer a dependency on synthetic biochemical building blocks for these proteins,” Isaacs told reporters during a conference call.

Both teams found that the cells perished in environments lacking the synthetic amino acid. Although the technology is not ready for industrial-scale deployment, the scientists suggested that such an approach could be applied as a safeguard against the escape of GMOs.

…..

 

‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment

Clement T Y ChanJeong Wook LeeD Ewen CameronCaleb J Bashor & James J Collins

Nature Chemical Biology(2015)            http://dx.doi.org:/10.1038/nchembio.1979

Figure 2: The fail-safe mechanism for Deadman circuit activation.

The fail-safe mechanism for Deadman circuit activation.

http://www.nature.com/nchembio/journal/vaop/ncurrent/carousel/nchembio.1979-F2.jpg

To demonstrate active control over host cell viability, cells grown under survival conditions (with ATc) were exposed to 1 mM IPTG to directly induce EcoRI and mf-Lon expression. Cell viability was measured by CFU count and is displayed…

 

Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the ‘Deadman’ and ‘Passcode’ kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently killEscherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

 

Nontoxic antimicrobials that evade drug resistance

Stephen A DavisBenjamin M VincentMatthew M EndoLuke WhitesellKaren MarchilloDavid R AndesSusan Lindquist & Martin D Burke

Nature Chemical Biology 2015;11:481–487          http://dx.doi.org:/10.1038/nchembio.1821

Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies.

 

 

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Genetically Engineered Algae

Curator: Larry H. Bernstein, MD, FCAP

 

 

Genetically engineered algae kill cancer cells

By Tim Sandle     Nov 11, 2015 in Science

http://www.digitaljournal.com/science/are-algae-the-solution-to-beating-cancer/article/449045#ixzz3rZQZ7pRj

A research group in Australia has developed algae nanoparticles. The algae have been found to kill 90 percent of cancer cells in cultured human cells. Based on this success, the modified algae have been shown to destroy cancerous tumors in mice.

 Algae is a general term for a range of single-celled microscopic photosynthetic organisms such as diatoms, and larger, multi-cellular plants, like kelp. The use of algae in cancer treatment relates to drug delivery. Some success has been achieved with the use of nanoparticles (such as nanoporous silica) to deliver anti-cancer medicines. One downside with this relates to the cost of production and the fact that some nanoparticles can be toxic. To overcome this, researchers based at the University of South Australia have used algae to model an alternative drug delivery system.
Success has been achieved with microalgae-derived nanoporous biosilica. The bio-material was derived from Thalassiosira pseudonana, which is a species of marine diatom. Diatoms are are among the most common types of phytoplankton, and they are found in the oceans, in freshwater, in soils and on damp surfaces. In studies, modified diatoms have been used as types of backpacks for the targeted delivery of anticancer drugs to tumor sites.

Lead researcher Nico Voelcker, speaking with International Business Times, explained why genetic modification was key in adapting the diatoms: “By genetically engineering diatom algae – tiny, unicellular, photosynthesising algae with a skeleton made of nanoporous silica, we are able to produce an antibody-binding protein on the surface of their shells. Anti-cancer chemotherapeutic drugs are often toxic to normal tissues.”

 Although the results are interesting, experiments in cell culture and even on mice do not necessarily translate into success in humans. A series of clinical trials will be required to verify the results and to streamline any potential treatment options.

The research is published in the journal Nature Communications, in a paper headed “Targeted drug delivery using genetically engineered diatom biosilica.”

Targeted drug delivery using genetically engineered diatom biosilica

 

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile ‘backpacks’ for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.

 

Figure 1: The principle of action of the genetically engineered biosilica therapeutic nanoparticles

The principle of action of the genetically engineered biosilica therapeutic nanoparticles.

http://www.nature.com/ncomms/2015/151110/ncomms9791/carousel/ncomms9791-f1.jpg

Genetically engineered diatom biosilica (green) containing liposome-encapsulated drug molecules (yellow) can be targeted to both adherent neuroblastoma cells (red) and lymphocyte cells in suspension (purple) by functionalizing the biosilica

 

Figure 2: SEM images of T. pseudonana biosilica and analysis of IgG–HRP binding to T. pseudonana biosilica

SEM images of T. pseudonana biosilica and analysis of IgG-HRP binding to T. pseudonana biosilica.

http://www.nature.com/ncomms/2015/151110/ncomms9791/carousel/ncomms9791-f2.jpg

(a) Side view and (b) top view of cylinder-shaped biosilica from a single diatom cell. (c,d) Details of the biosilica structure showing the highly porous surface. (e) Schematic structure of the S–T8–GFP–GB1 fusion protein. S, N-terminal…

 

Figure 4: Interaction of anti-CD20 antibody-labelled GB1–biosilica with B and T cells captured on an anti-CD45 antibody microarray.

Interaction of anti-CD20 antibody-labelled GB1-biosilica with B and T cells captured on an anti-CD45 antibody microarray.

http://www.nature.com/ncomms/2015/151110/ncomms9791/carousel/ncomms9791-f4.jpg

The left panels show epifluorescence microscopy images of cells captured on microarray spots after incubation with anti-CD20 antibody-labelled biosilica (n=3). The right panels show higher magnification images within the spots. Green: b…

 

Figure 6: Interaction of anti-p75NTR–GB1–biosilica frustules with adherent cells

Interaction of anti-p75NTR-GB1-biosilica frustules with adherent cells.

http://www.nature.com/ncomms/2015/151110/ncomms9791/carousel/ncomms9791-f6.jpg

(a) Confocal fluorescence microscopy image of SH-SY5Y neuroblastoma cells and (b) BSR fibroblast cells (n=3). Red: actin (phalloidin staining, green: biosilica (GFP-labelled), blue: nucleus (Hoechst 33342 staining). (c) The left panel s…

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Gene Editing by creation of a complement without transcription error

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Nanoparticle-Based Artificial Transcription Factor  

NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation

Abstract Image

Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is a promising approach for many biological applications; however, several limitations hinder the full potential of TFs. Herein, we developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. We demonstrate that NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by over 15-fold. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner. Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential for various stem cell applications.

NanoScript_emulates_TF_Structure_and_Function_large.jpg

http://www.energyigert.rutgers.edu/sites/default/files/faculty/kibumlee/NanoScript_emulates_TF_Structure_and_Function_large.jpg

HIGHLIGHTS

  • Transcription Factors (TF) are proteins that regulate transcription and gene expression
  • NanoScript is an versatile, nanoparticle-based platform that mimics TF structure and biological function
  • NanoScript is stable in physiological environments and localizes within the nucleus
  • NanoScript initiates targeted gene expression by over 15-fold to 30 fold, which would be critical for stem cell differentiation and cellular reprogramming
  • NanoScript transcribes endogenous genes on native DNA in a non-viral manner

Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is an essential approach for many biological applications such as stem cell differentiation and cellular programming, however, several limitations hinder the full potential of TFs.

To address this challenge, researchers in Prof. KiBum Lee’s group (Sahishnu Patel and Perry Yin) developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. They demonstrated that NanoScript localizes within the nucleus and initiates transcription of a targeted gene with high efficiency. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a non-viral manner.

NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform. NanoScript has two attractive features that make this the perfect platform for stem cell-based application. First, because gene regulation by NanoScript is non-viral, it serves as an attractive alternative to current differentiation methods that use viral vectors. Second, by simply rearranging the sequence of one molecule on NanoScript, NanoScript can target any differentiation-specific genes and induce differentiation, and thus has excellent prospect for applications in stem cell biology and cellular reprogramming.

Perry To-tien Yin
PhD Candidate, Rutgers University
Prospects for graphene–nanoparticle-based hybrid sensors

PT Yin, TH Kim, JW Choi, KB Lee
Physical Chemistry Chemical Physics 15 (31), 12785-12799
31 2013
Axonal Alignment and Enhanced Neuronal Differentiation of Neural Stem Cells on Graphene‐Nanoparticle Hybrid Structures

A Solanki, STD Chueng, PT Yin, R Kappera, M Chhowalla, KB Lee
Advanced Materials 25 (38), 5477-5482
22 2013
Label‐Free Polypeptide‐Based Enzyme Detection Using a Graphene‐Nanoparticle Hybrid Sensor

S Myung, PT Yin, C Kim, J Park, A Solanki, PI Reyes, Y Lu, KS Kim, …
Advanced Materials 24 (45), 6081-6087
22 2012
Guiding Stem Cell Differentiation into Oligodendrocytes Using Graphene‐Nanofiber Hybrid Scaffolds

S Shah, PT Yin, TM Uehara, STD Chueng, L Yang, KB Lee
Advanced materials 26 (22), 3673-3680
21 2014
Design, Synthesis, and Characterization of Graphene–Nanoparticle Hybrid Materials for Bioapplications

PT Yin, S Shah, M Chhowalla, KB Lee
Chemical reviews 115 (7), 2483-2531
16 2015
Multimodal Magnetic Core–Shell Nanoparticles for Effective Stem‐Cell Differentiation and Imaging

B Shah, PT Yin, S Ghoshal, KB Lee
Angewandte Chemie 125 (24), 6310-6315
16 2013
Nanotopography-mediated reverse uptake for siRNA delivery into neural stem cells to enhance neuronal differentiation

A Solanki, S Shah, PT Yin, KB Lee
Scientific reports 3
14 2013
Combined Magnetic Nanoparticle‐based MicroRNA and Hyperthermia Therapy to Enhance Apoptosis in Brain Cancer Cells

PT Yin, BP Shah, KB Lee
small 10 (20), 4106-4112
11 2014

A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics

(Nanowerk News) Associate Professor Ki-Bum Lee has developed patent-pending technology that may overcome one of the critical barriers to harnessing the full therapeutic potential of stem cells.
One of the major challenges facing researchers interested in regenerating cells and growing new tissue to treat debilitating injuries and diseases such as Parkinson’s disease, heart disease, and spinal cord trauma, is creating an easy, effective, and non-toxic methodology to control differentiation into specific cell lineages. Lee and colleagues at Rutgers and Kyoto University in Japan have invented a platform they call NanoScript, an important breakthrough for researchers in the area of gene expression. Gene expression is the way information encoded in a gene is used to direct the assembly of a protein molecule, which is integral to the process of tissue development through stem cell therapeutics.
Stem cells hold great promise for a wide range of medical therapeutics as they have the ability to grow tissue throughout the body. In many tissues, stem cells have an almost limitless ability to divide and replenish other cells, serving as an internal repair system.
Nanoscript

Schematic representation of NanoScript’s design and function. (a) By assembling individual STF molecules, including the DBD (DNA-binding domain), AD (activation domain), and NLS (nuclear localization signal), onto a single 10 nm gold nanoparticle, we have developed the NanoScript platform to replicate the structure and function of TFs. This NanoScript penetrates the cell membrane and enters the nucleus through the nuclear receptor with the help of the NLS peptide. Once in the nucleus, NanoScript interacts with DNA to initiate transcriptional activity and induce gene expression. (b) When comparing the structure of NanoScript to representative TF proteins, the three essential domains are effectively replicated. The linker domain (LD) fuses the multidomain protein together and is replicated by the gold nanoparticle (AuNP). (c) The DBD binds to complementary DNA sequences, while the AD recruits transcriptional machinery components such as RNA polymerase II (RNA Pol II), mediator complex, and general transcription factors (GTFs). The synergistic function of the DBD and AD moieties on NanoScript initiates transcriptional activity and expression of targeted genes. (d) The AuNPs are monodisperse and uniform. The NanoScript constructs are shown to effectively localize within the nucleus, which is important because transcriptional activity occurs only in the nucleus. (Reprinted with permission y American Chemical Society) (click on image to enlarge)

Read more: Using nanotechnology to regulate gene expression at the transcriptional level

Transcription factor (TF) proteins are master regulators of gene expression. TF proteins play a pivotal role in regulating stem cell differentiation. Although some have tried to make synthetic molecules that perform the functions of natural transcription factors, NanoScript is the first nanomaterial TF protein that can interact with endogenous DNA.
ACS Nano, a publication of the American Chemical Society (ACS), has published Lee’s research on NanoScript (“NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation”). The research is supported by a grant from the National Institutes of Health (NIH).
“Our motivation was to develop a highly robust, efficient nanoparticle-based platform that can regulate gene expression and eventually stem cell differentiation,” said Lee, who leads a Rutgers research group primarily focused on developing and integrating nanotechnology with chemical biology to modulate signaling pathways in cancer and stem cells. “Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential to do exactly that. The field of stem cell biology now has another platform to regulate differentiation while the field of nanotechnology has demonstrated for the first time that we can regulate gene expression at the transcriptional level.”
NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles.
“NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by up to 30-fold,” said Sahishnu Patel, Rutgers Chemistry graduate student and co-author of the ACS Nano publication. “NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner.”
Lee said the next step for his research is to study what happens to the gold nanoparticles after NanoScript is utilized, to ensure no toxic effects arise, and to ensure the effectiveness of NanoScript over long periods of time.
“Due to the unique tunable properties of NanoScript, we are highly confident this platform not only will serve as a desirable alternative to conventional gene-regulating methods,” Lee said, “but also has direct employment for applications involving gene manipulation such as stem cell differentiation, cancer therapy, and cellular reprogramming. Our research will continue to evaluate the long-term implications for the technology.”
Lee, originally from South Korea, joined the Rutgers faculty in 2008 and has earned many honors including the NIH Director’s New Innovator Award. Lee received his Ph.D. in Chemistry from Northwestern University where he studied with Professor Chad. A. Mirkin, a pioneer in the coupling of nanotechnology and biomolecules. Lee completed his postdoctoral training at The Scripps Research Institute with Professor Peter G. Schultz. Lee has served as a Visiting Scholar at both Princeton University and UCLA Medical School.
The primary interest of Lee’s group is to develop and integrate nanotechnologies and chemical functional genomics to modulate signaling pathways in mammalian cells towards specific cell lineages or behaviors. He has published more than 50 articles and filed for 17 corresponding patents.
Source: Rutgers University

Read more: A highly robust, efficient nanoparticle-based platform to advance stem cell therapeutics

Nanoparticle-based transcription factor mimics

http://nanomedicine.ucsd.edu/blog/article/nanoparticle-based-transcription-factor-mimics

Biologists have been enhancing expression of specific genes with plasmids and viruses for decades, which has been essential to uncovering the function of numerous genes and the relationships among the proteins they encode. However, tools that allow enhancement of expression of endogenous genes at the transcriptional level could be a powerful complement to these strategies. Many chemical biologists have made enormous progress developing molecular tools for this purpose; recent work by a group at Rutgers suggests how nanotechnology might allow application of this strategy in living organisms, and perhaps one day in patients.

In a paper published in ACS Nano, researchers led by KiBum Lee synthesized gold nanoparticles bearing synthetic or shortened versions of the three essential components of transcription factors (TFs), the proteins that “turn on” expression of specific genes in cells. Specifically, polyamides previously designed to bind to a specific promoter sequence, transactivation peptides, and nuclear localization peptides were conjugated to the nanoparticle surface. These nanoparticles enhanced expression of both a reporter plasmid (by ~15-fold) and several endogenous genes (by up to 65%). This enhancement is much greater than that possible using previous constructs lacking nuclear localization sequences; the team incorporated a high proportion of those peptides to ensure efficient delivery to the nucleus.

Nanoscript, a synthetic transciption factor
Diagram of the synthetic TF mimic (termed NanoScript). Decorated particles are ~35 nm in diameter. Letters are amino acid sequences; Py-Im, N-methylpyrrole-N-methylimidazole.

These nanoparticles offer an alternative to delivering protein TFs, which remains extremely challenging despite considerable effort towards the development of delivery systems that transport cargo into cells. Among other barriers to the use of native TFs, incorporating them into polymeric or lipid-based carriers often alters their shape, which would likely reduce their function.

While the group suggests future generations of these nanoparticles might one day be used to treat diseases caused by defects in TF genes, many questions remain. First, the duration of gene expression enhancement is not known; the study only assesses effects at 48 h post-administration. Further, whether gold is the best material for the core remains unclear, as its non-biodegradability means the particles would likely accumulate in the liver over time; synthetic TFs with biodegradable cores might also be considered.

Patel S et al., NanoScript: a nanoparticle-based artificial transcription factor for effective gene regulation,ACS Nano 2014; published online Sep 3.

http://www.wtec.org/bem/docs/BEM-FinalReport-Web.pdf

Biocompatibility and Toxicity of Nanobiomaterials

“Biocompatibility and Toxicity of Nanobiomaterials” is an annual special issue published in “Journal of Nanomaterials.”

http://www.hindawi.com/journals/jnm/toxicity.nanobiomaterials/

Porous Ti6Al4V Scaffold Directly Fabricated by Sintering: Preparation and In Vivo Experiment
Xuesong Zhang, Guoquan Zheng, Jiaqi Wang, Yonggang Zhang, Guoqiang Zhang, Zhongli Li, and Yan Wang
Department of Orthopaedics, Chinese People’s Liberation Army General Hospital, Beijing 100853, China AcademicEditor:XiaomingLi
The interface between the implant and host bone plays a key role in maintaining primary and long-term stability of the implants. Surface modification of implant can enhance bone in growth and increase bone formation to create firm osseo integration between the implant and host bone and reduce the risk of implant losing. This paper mainly focuses on the fabricating of 3-dimensiona interconnected porous titanium by sintering of Ti6Al4V powders, which could be processed to the surface of the implant shaft and was integrated with bone morphogenetic proteins (BMPs). The structure and mechanical property of porous Ti6Al4V was observed and tested. Implant shaft with surface of porous titanium was implanted into the femoral medullary cavity of dog after combining with BMPs. The results showed that the structure and elastic modulus of 3D interconnected porous titanium was similar to cancellous bone; porous titanium combined with BMP was found to have large amount of fibrous tissue with fibroblastic cells; bone formation was significantly greater in 6 weeks postoperatively than in 3 weeks after operation. Porous titanium fabricated by powders sintering and combined with BMPs could induce tissue formation and increase bone formation to create firm osseo integration between the implant and host bone.

Journal of Materials Chemistry B   Issue 39, 2013

Materials for biology and medicine
Synthesis of nanoparticles, their biocompatibility, and toxicity behavior for biomedical applications
J. Mater. Chem. B, 2013,1, 5186-5200    DOI: http://dx.doi.org:/10.1039/C3TB20738B

Nanomaterials research has in part been focused on their use in biomedical applications for more than several decades. However, in recent years this field has been developing to a much more advanced stage by carefully controlling the size, shape, and surface-modification of nanoparticles. This review provides an overview of two classes of nanoparticles, namely iron oxide and NaLnF4, and synthesis methods, characterization techniques, study of biocompatibility, toxicity behavior, and applications of iron oxide nanoparticles and NaLnF4nanoparticles as contrast agents in magnetic resonance imaging. Their optical properties will only briefly be mentioned. Iron oxide nanoparticles show a saturation of magnetization at low field, therefore, the focus will be MLnF4 (Ln = Dy3+, Ho3+, and Gd3+) paramagnetic nanoparticles as alternative contrast agents which can sustain their magnetization at high field. The reason is that more potent contrast agents are needed at magnetic fields higher than 7 T, where most animal MRI is being done these days. Furthermore we observe that the extent of cytotoxicity is not fully understood at present, in part because it is dependent on the size, capping materials, dose of nanoparticles, and surface chemistry, and thus needs optimization of the multidimensional phenomenon. Therefore, it needs further careful investigation before being used in clinical applications.

Graphical abstract: Synthesis of nanoparticles, their biocompatibility, and toxicity behavior for biomedical applications

http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=C3TB20738B

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citations…

HAMLET interacts with lipid membranes and perturbs their structure and integrity

HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal …. of the alternative complement pathway preserves photoreceptors after retinal injury ….. Life-long in vivo cell-lineage tracing shows that no oogenesis originates from …. ananoparticle-based artificial transcription factor for effective gene regulation …

Authors: Ann-Kristin Mossberg, Maja Puchades, Øyvind Halskau, Anne Baumann, Ingela Lanekoff, Yinxia Chao, Aurora Martinez, Catharina Svanborg, & Roger Karlsson

www.regenerativemedicine.net/NewsletterArchives.asp?qEmpID…

Summary: 

Background – Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.

Methodology/Principal Findings – We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.

Conclusions/Significance – The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

Source: Public Library of Science ONE; 5(2) (02/23/10) 

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