Posts Tagged ‘Massachusetts Institute of Technology’

Reporter: Prabodh Kandala, PhD

A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.

But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.

The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.

Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”

Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.

Power struggle

Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.

Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.

To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.

They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”

This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.

“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”

When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.

Tipping the balance

In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.

The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.

Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.

In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.


Massachusetts Institute of Technology (2013, February 4). Some cancer mutations slow tumor growth. ScienceDaily. Retrieved February 4, 2013, from http://www.sciencedaily.com­/releases/2013/02/130204154011.htm

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Reporter: Aviva Lev-Ari, PhD, RN


Ashley Yeager

For the first time, scientists have identified all the proteins in the mitochondrial matrix, which is where the cell’s energy is generated. How? Find out…


Between the maze-like inner membranes of the mitochondria, there’s a thick, sticky region called the matrix. This region serves an important role in the generation of the cell’s energy, but the proteins that actually make up this matrix have remained a mystery. But now, researchers at the Massachusetts Institute of Technology (MIT) have catalogued all the proteins in the mitochondrial matrix, identifying 31 proteins not previously associated with mitochondria. They did this by combining the strengths of two methods: microscopy and mass spectrometry.


Electron microscopy of human embryonic kidney cells expressing mito-APEX. Credit: Alice Ting, MIT

“This method is really a new paradigm for doing mass spec proteomics because we’re recording proteomics in living cells,” said Alice Ting, a chemist at MIT and author of a paper published online yesterday in Science that describes the technique (1).Microscopy and mass spectrometry are valuable for studying proteins, but each has its drawbacks. While microscopy can show where a protein is located within a cell, it can only do so for a small number of a cell’s roughly 20,000 proteins at once. Meanwhile, mass spectrometry can identify all the proteins within a cell, but destroys the cell membrane in the process of releasing the cell’s contents, resulting in a mixture of proteins from different cell regions and organelles.

To overcome these limitations, Ting’s group genetically engineered the mitochondrial matrix to express a newly designed peroxidase called APEX. When biotin-phenol was added to these cells, APEX stripped an electron and a proton from the biotin molecule, creating highly reactive biotin-phenoxyl radicals. These radicals quickly bound to nearby proteins to stabilize themselves, effectively tagging the proteins in the matrix.

The scientists then identified these tagged proteins with fluorescent imaging, dissolved the cell membrane, and isolated the proteins from the mitochondrial matrix. Using mass spectrometry, the team then identified 495 proteins in the mitochondrial matrix, 31 of which had not been previously linked to the mitochondrial region.

One of the biggest surprises was the discovery that the enzyme PPOX is in the matrix. PPOX helps synthesize heme, the pigment in red blood cells and a cofactor of the protein hemoglobin. Previously, biologists believed that PPOX was located within the space between the outer and inner membranes of the mitochondria, but Ting’s team found that it was actually within the matrix, which the team said is an example of how locally precise their biotin-tagging technique is.

Now, Ting and her team are looking at proteins in the mitochondrial intermembrane space. In addition, the researchers are tweaking their labeling system to map proteins in the cell membrane and to detect specific protein-protein interactions.


1. Rhee, H.-W., P. Zou, N. D. Udeshi, J. D. Martell, V. K. Mootha, S. A. Carr, and A. Y. Ting. 2013. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science (January).




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Reporter: Aviva Lev-Ari, RN

With New Microfludic Technique, MIT Team Aims to ‘Squeeze’ siRNAs into Cells

January 31, 2013

Researchers from the Massachusetts Institute of Technology last week reported on the development of a new microfluidic-based approach to delivering macromolecules, including functional siRNAs, into cells without the need for a vector.

According to the investigators, who published their findings in the Proceedings of the National Academy of Sciences, the technique involves compressing cells by passing them through a constriction, which opens up temporary holes in their membranes that permit the diffusion of materials in surrounding buffer to enter the cytosol.

“By providing flexibility in application and obviating the need for exogenous materials or electrical fields, this method could potentially enable new avenues of disease research and treatment,” they wrote.

Although intracellular delivery of macromolecules is a key step in therapeutic and research applications, the cellular membrane is largely impermeable to such compounds, according to the PNAS paper. Existing methods to overcome this hurdle, which has proven to be a major stumbling block for RNAi drugs, typically involve the use of polymeric nanoparticles, liposomes, or chemical modifications of the target molecules to facilitate membrane poration or endocytotic delivery.

When it comes to nucleic acids, which are relatively structurally uniform, these approaches can be efficient. Still, the “endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles,” the MIT team pointed out. “More effective gene delivery methods, such as viral vectors, however, often risk chromosomal integration.

Meantime, electroporation has proven effective, even in difficult to transfect primary cells, but has limited applicability and can cause cell death. Microinjection, too, has certain advantages in settings such as the creation of transgenic organisms, but its low throughput hamstrings many therapeutic and research applications, the researchers noted.

To overcome the limitations of existing delivery techniques, the MIT group had initially been attempting to “shoot” molecules of interest into cells, Armon Sharei, an MIT graduate student in chemical engineering and lead author of the PNAS paper, told Gene Silencing News.

“That system had its own challenges, and through the course of that work, we stumbled upon this effect where if you squeeze the cells rapidly enough, it will temporarily disrupt their membrane,” he said.

More specifically, the researchers found that the “rapid mechanical deformation of a cell, as it passes through a constriction with a minimum dimension smaller than the cell diameter, results in the formation of transient membrane disruptions or holes,” they wrote in PNAS. “The size and frequency of these holes would be a function of the shear and compressive forces experienced by the cell during its passage through the constriction. Material from the surrounding medium may then diffuse directly into the cell cytosol throughout the life span of these holes.”

To test this idea, the researchers constructed devices, each consisting of 45 identical, parallel microfluidic channels containing one or more constrictions, etched onto a silicon chip and sealed in glass. The width of each constriction ranged from 4 to 8 micrometers, and the lengths ranged from 10 to 40 micrometers.

“Before use, the device is first connected to a steel interface that connects the inlet and outlet reservoirs to the silicon device,” the researchers wrote. “A mixture of cells and the desired delivery material is then placed into the inlet reservoir and Teflon tubing is attached at the inlet. A pressure regulator is then used to adjust the pressure at the inlet reservoir and drive the cells through the device. Treated cells are collected from the outlet reservoir.”

The system was tested with a variety of molecules, including carbon nanotubes and proteins, as well as siRNAs targeting GFP. According to Sharei, when the siRNAs were delivered into GFP-expressing HeLa cells using the microfluidic platform, the investigators were able to achieve 80 to 90 percent target knockdown.

He noted that the knockdown effects weren’t as robust as with Lipofectamine 2000, but “we were still encouraged because something like Lipofectamine is known to be toxic and therefore inapplicable for humans.” Notably, the microfluidic device and operating parameters were not optimized for siRNAs, further limiting its ability to compete with the transfection reagent in these studies.

“The other good thing was that we seem to work just as well for primary cells, whereas existing methods like Lipofectamine don’t translate well once you start moving out of the standard cell models you have in the lab,” he added.

The MIT team also successfully delivered 3 kilodalton dextran molecules — which are approximately the same size as a standard siRNA molecule and a “pretty accurate” surrogate for the gene-silencing molecules — into newborn human foreskin fibroblasts, primary murine dendritic cells, and embryonic stem cells, suggesting that the method could be used with siRNAs into a variety of cell types, Sharei said.

Buoyed by the positive data, he and his colleagues are now further testing the platform with siRNAs against “easy readout genes” in primary cells including immune cells and stem cells, he said. “Once we establish that, we’d try to go for an application where there’s an siRNA that’s going to knock down something functional.

“I can’t say exactly what we’ve been up to because it’s not published, but it has been going pretty well,” he added.

Ultimately, the MIT group aims to develop the microfluidic platform not only for research applications, where it could be “incorporated into a larger integrated system consisting of multiple pre-treatment and post-treatment modules” that could take advantage of its average throughput rate of 20,000 cells a second, but also therapeutic ones, too.

A number of investigational stem cell-based therapies, for instance, involve the ex vivo manipulation of the cells, Sharei said. The delivery platform could theoretically be used to “enhance or facilitate that manipulation.”

“Such an approach would take advantage of the potentially increased delivery efficiency of therapeutic macro- molecules and could be safer than existing techniques because it would obviate the need for potentially toxic vector particles and would mitigate any potential side effects associated with reticuloendothelial clearance and off-target delivery,” the study authors wrote in PNAS.

Doug Macron is the editor of GenomeWeb’s Gene Silencing News. He covers research and therapeutic applications of RNAi, miRNA, and other gene-silencing technologies. E-mail Doug Macron or follow his GenomeWeb Twitter account at@Genesilencing.


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Reporter: Aviva Lev-Ari, PhD, RN

On August 18, 2012, I needed scientific inspiration. To nurture my imagination I surf on http://www.mit.edu for 4 hours. Comment, Please.

The following links I am sharing with you from my exploration session to be inspired

Protein that boosts longevity may protect against diabetes: Sirtuins help fight off disorders linked to obesity, new MIT study shows.


Growing the best implant tissue | MIT video


MIT 2012 Commencement Address


Salman Khan talk at TED 2011 (from ted.com)



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Cello Music Concert by Jacqueline du Pre


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