MIT engineers have developed a new, highly efficient way to pair up cells so they can be fused together into a hybrid cell.

The new technique should make it much easier for scientists to study what happens when two cells are combined.  For example, fusing an adult cell and an embryonic stem cell allows researchers to study the genetic reprogramming that occurs in such hybrids.

The researchers, led by a collaboration between Joel Voldman, associate professor of electrical engineering and computer science, and Rudolf Jaenisch, professor of biology and a member of the Whitehead Institute, report the new technique in the Jan. 4 online edition of Nature Methods.

The work was spearheaded by two postdoctoral associates, Alison Skelley, who worked in Voldman’s lab, and Oktay Kirak, who works with Jaenisch.  Skelley and Kirak are lead authors of the Nature Methods paper.  Heikyung Suh, a technical associate in the Whitehead Institute, is also an author of the paper.

The team’s simple but ingenious sorting method increases the rate of successful cell fusion from around 10 percent to about 50 percent, and allows thousands of cell pairings at once.

Though cell fusion techniques have been around for a long time, there are many technical limitations, said Voldman.

Getting the right cells to pair up before fusing them is one major obstacle.  If scientists are working with a mixture of two cell types, for example A and B, they end up with many AA and BB pairings, as well as the desired AB match.

Researchers had previously trapped cells in tiny cups as they flow across a chip.  Each cup can hold only two cells, but there is no way to control whether the cups capture an A and a B, two As or two Bs.

In contrast, the cell-trapping cups on Voldman and Jaenisch’s new sorting device are arranged strategically to capture and pair up cells of different types.

First, type A cells are flowed across the chip in one direction and caught in traps that are large enough to hold only one cell.  Once the cells are trapped, liquid is flowed across the chip in the opposite direction, pushing the cells out of the small cups and into larger cups across from the small ones.

Once one A cell is in each large cup, type B cells are flowed into the large cups.  Each cup can only hold two cells, so each ends up with one A and one B. After the cells are paired in the traps, they can be joined together by an electric pulse that fuses the cell membranes.

In addition to helping with studies of stem cell reprogramming, this technique could be used to study interactions between any types of cells.  “It’s a very general type of device,” said Voldman.

In the tiny realm of nanotechnology, scientists have used a wide variety of materials to build atomic scale structures.  But just as in the construction business, nanotechnology researchers can often be limited by the amount of raw materials.  Now, Biodesign Institute at Arizona State University researcher Hao Yan has avoided these pitfalls by using cells as factories to make DNA based nanostructures inside a living cell.

The results were published in the early online edition of the Proceedings of the National Academy of Sciences.

Yan specializes in a fast-growing field within nanotechnology -commonly known as structural DNA nanotechnology -that uses the basic chemical units of DNA, abbreviated as C, T, A, or G, to self-fold into a number of different building blocks that can further self-assemble into patterned structures.

“This is a good example of artificial nanostructures that can be replicated using the machineries in live cells” said Yan.  “Cells are really good at making copies of double stranded DNA and we have used the cell like a copier machine to produce many, many copies of complex DNA nanostructures.”

DNA nanotechnologists have made some very exciting achievements during the past five to 10 years.  But DNA nanotechnology has been limited by the need to chemically synthesize all of the material from scratch.  To date, it has strictly been a test tube science, where researchers have developed many toolboxes for making different DNA nanostructures to attach and organize other molecules including nanoparticles and other biomolecules.

“If you need to make a single gram of a DNA nanostructure, you need to order one gram of the starting DNA materials.  Scientists have previously used chemical methods to copy branched DNA structures, and there has also been significant work in using long-stranded DNA sequences replicated from cells or phage viruses to scaffold short helper DNA sequences to form 2-D or 3-D objects,” said Yan, who is also a professor in the Department of Chemistry and Biochemistry at ASU.

“We have always dreamed of scaling up DNA nanotechnology.  One way to scale that it up is to use the cellular system because simple DNA can be replicated inside the cell.  We wanted to know if the cell’s copy machine could tolerate single stranded DNA nanostructures that contain complicated secondary structures.”

To test the nanoscale manufacturing capabilities of cells, Yan and his fellow researchers, Chenxiang Lin, Sherri Rinker and Yan Liu at ASU and their collaborators Ned Seeman and Xing Wang at New York University went back to reproducing the very first branched nanostructure made up of DNAa cross-shaped, four-arm DNA junction and another DNA junction structure containing a different crossover topology.

To copy these branched DNA nanostructures inside a living cell, the ASU and NYU research team first shipped the cargo inside a bacteria cell.  They cut and pasted the DNA necessary to make these structures into a phagemid, a virus-like particle that infects a bacteria cell.  Once inside the cell, the phagemid used the cell just like a photocopier machine to reproduce millions of copies of the DNA.  By theoretically starting with just a single phagemid infection, and a single milliliter of cultured cells, Yan found that the cells could churn out trillions of the DNA junction nanostructures.

The DNA nanostructures produced in the cells were also found to fold correctly, just like the previously built test tube structures.  According to Yan, the results also proved the key existence of the DNA nanostructures during the cell’s routine DNA replication and division cycles.  “When a DNA nanostructure gets replicated, it does exist and can survive the complicated cellular machinery.  And it looks like the cell can tolerate this kind of structure and still do its job.  It’s amazing,” said Yan.

Yan acknowledges that this is just the first step, but foresees there are many interesting DNA variations to consider next.  “The fact that the natural cellular machinery can tolerate artificial DNA objects is quite intriguing, and we don’t know what the limit is yet.”

Yan’s group may be able to change and evolve DNA nanostructures and devices using the cellular system and the technology may also open up some possibilities for synthetic biology applications.

“I’m very excited about the future of DNA nanotechnology, but there is a lot of work to be done.  An interesting research topic to pursue is the interface of DNA nanostructures with live cells; it is full of opportunities,” said Yan.

Antibodies are proteins that are a crucial component of the immune system.  They are produced in large amounts by immune cells known as plasma cells, which live in just a few parts of the body, including the bone marrow and special areas of the various parts of the body that are exposed to the outside (e.g., the gut, nose, and airways).  These areas are known as mucosa-associated lymphoid tissue (MALT) and include tissues such as the tonsils, but what regulates plasma cell survival in MALT has not been determined.  Now, however, Bertrand Huard and colleagues, at Geneva University Medical Center, Switzerland, have provided new insight into the molecular mechanisms controlling plasma cell survival in MALT.

In the study, analysis of tonsils and MALT from the lower gut indicated that a protein known as APRIL is important for promoting the survival of plasma cells in human MALT.  APRIL was found to work by increasing plasma cell expression of proteins that protect cells from a form of death known as apoptosis.  Expression of APRIL was shown to be greater in tonsils infected with a microbe than in noninfected tonsils and the cells producing the increased APRIL were identified as immune cells known as neutrophils that had been recruited to the site of infection.  APRIL from the neutrophils was retained in the tonsils bound to molecules known as heparan sulfate proteoglycans, creating an APRIL-rich niche for the plasma cells to survive in.  The authors therefore suggest that the longevity of plasma cells in MALT is controlled, in part, by APRIL-secreting neutrophils recruited to sites of infection.

Although prostate cancer is the second most common cause of death from cancer in the US, it is not the tumor in the prostate that usually causes death.  Rather, death mainly occurs as a result of the tumor spreading to the bones, where it is known as an osteoblastic bone metastasis.  Treatments that deprive the tumor of male sex hormones (androgens) are usually effective, but only briefly as the tumors typically develop the ability to grow in the absence of androgens and the diseases progresses.  New data, generated using two prostate cancer cell lines that lack expression of androgen receptors and that were derived from the bones of an individual with osteoblastic bone metastases, by Nora Navone and colleagues, at The University of Texas MD Anderson Cancer Center, Houston, have provided new insight into the mechanisms by which prostate cancer osteoblastic bone metastases progress.

The androgen receptor–negative prostate cancer cell lines generated by the authors grew when transplanted into immunocompromised mice and generated osteoblastic bone metastases.  A protein known as FGF9 was found to be expressed at higher levels in these cells lines than in other bone-derived prostate cancer cells and induced bone formation in an in vitro organ culture assay.  Further, as blocking FGF9 reduced the osteoblastic bone metastases in mice transplanted with the cell lines and FGF9 was found to be expressed in all human prostate cancer osteoblastic bone metastases analyzed, the authors suggest that FGF9 has an important role in prostate cancer progression to osteoblastic bone metastases.  The cells lines generated are also likely to be an important preclinical model for researchers developing therapeutics for osteoblastic bone metastases in individuals with prostate cancer.

Switches are a part of daily life, from snoozing your alarm, turning on the coffee maker, firing up your car engine, and so on until we turn off the lights at night. Researchers have now cataloged even more templates of possible switches within a living cell than we use throughout our day.Naren Ramakrishnan, associate professor of computer science at Virginia Tech, USA, and Upinder S. Bhalla, at the National Centre for Biological Sciences (NCBS), part of the Tata Institute of Fundamental Research in India, found that cells can make use of thousands of switches to support important biological functions.

Cells use switches for determining what kind of cell to become – skin or blood, for instance, in responding to stress, and in communication with other cells. “A switch is like a memory unit,” said Bhalla. “The state of the switch — whether it is on or off, is like a computer memory that can store a bit of 0 or 1. Although real biological switches are quite complex and regulated in many ways, we have shown the simplest possible ways in which switches could work”, Bhalla said.

The researchers report their work in the June 20 issue of the Public Library of Science (PLoS) Computational Biology, in the article “Memory Switches in Chemical Reaction Space.” Their collaboration began during a sabbatical visit by Ramakrishnan to NCBS in Bangalore, India. Ramakrishnan is a computer scientist whose expertise is in numerical simulation and data mining. Bhalla is a computational neuroscientist with broad interests in biochemical network modeling and simulation. They decided to use Virginia Tech’s System X supercomputer to search for the many ways in which cells can implement switches.

“Our exploration using System X is rather like how a tinkerer or a kid puts together things to see if they do something useful. We took a lot of ’spare parts’, each spare part being one chemical reaction, connected them together every which way, and we found that a surprising number of these artificially constructed networks actually were switches,” said Ramakrishnan.

“Popular opinion used to be that there are a small number of ways in which switches can be realized by biology, but we found thousands of switches in our search,” Ramakrishnan said.

The researchers report in PLoS Computational Biology, “We find nearly 4,500 reaction topologies, or about 10 percent of our tested configurations, that demonstrate switching behavior.”

Their research also led to a comprehensive “map” of biochemical switches. The map further revealed that most of the switches form a “family” – that is, the switches are all related to one another. “This has important implications since it suggests how evolution might stumble upon a switch rather easily.” Ramakrishnan said.

“Of course, there is more to cells than switches,” Bhalla said. “But switching and memory are the most basic behaviors possible. Armed with our catalog of switches, we can now proceed to investigate more interesting behaviors like complex information processing.”

As tumors progress they develop ways to escape recognition and attack by cells of the immune system. However, the mechanisms by which tumors modify the immune system have not been clearly determined. New insight into the way in which chronic lymphocytic leukemia (CLL) cells limit immune cell attack has now been provided by John Gribben and colleagues, at Barts and The London School of Medicine, United Kingdom.

For immune cells known as CD4+ and CD8+ T cells to become activated they must contact other cells known as APCs. The area of contact is known as the immunological synapse and it is highly organized. In the study, CD4+ and CD8+ T cells from patients with CLL were found to exhibit defective immunological synapse formation with APCs. Further, if CD4+ and CD8+ T cells from healthy individuals were cultured with CLL APCs, they also showed defective immunological synapse formation. As treatment with an immune system–modifying drug improved immunological synapse formation, the authors suggest that approaches to overcoming immunological synapse defects might improve the efficacy of new ways to treat cancer that are currently being developed and that are based on enhancing the antitumor activity of CD4+ and CD8+ T cells.

Researchers have uncovered details about how dietary restriction slows down aging. A team of University of Washington scientists have uncovered details about the mechanisms through which dietary restriction slows the aging process.  Working in yeast cells, the researchers have linked ribosomes, the protein-making factories in living cells, and Gcn4, a specialized protein that aids in the expression of genetic information, to the pathways related to dietary response and aging.  The study, which was led by UW faculty members Brian Kennedy and Matt Kaeberlein, appears in the April 18 issue of the journal Cell.

Previous research has shown that the lifespan-extending properties of dietary restriction are mediated in part by reduced signaling through TOR, an enzyme involved in many vital operations in a cell.  When an organism has less TOR signaling in response to dietary restriction, one side effect is that the organism also decreases the rate at which it makes new proteins, a process called translation.

In this project, the UW researchers studied many different strains of yeast cells that had lower protein production.  They found that mutations to the ribosome, the cell’s protein factory, sometimes led to increased life span.  Ribosomes are made up of two parts -the large and small subunits -and the researchers tried to isolate the life-span-related mutation to one of those parts.

“What we noticed right away was that the long-lived strains always had mutations in the large ribosomal subunit and never in the small subunit,” said the study’s lead author, Kristan Steffen, a graduate student in the UW Department of Biochemistry.

The researchers also tested a drug called diazaborine, which specifically interferes with synthesis of the ribosomes’ large subunits, but not small subunits, and found that treating cells with the drug made them live about 50 percent longer than untreated cells.  Using a series of genetic tests, the scientists then showed that depletion of the ribosomes’ large subunits was likely to be increasing life span by a mechanism related to dietary restriction -the TOR signaling pathway.

“We knew that dietary restriction decreased TOR signaling, and that decreased TOR signaling reduced translation or protein production, but this was the first direct evidence that all three were acting in the same genetic pathway,” said Kennedy, an associate professor of biochemistry.

“The big question then became what’s happening in these translation-deficient cells to slow aging,” added Kaeberlein, an assistant professor of pathology.  “That’s when Vivian MacKay, a co-author on the study, had the idea to look at Gcn4.”

Gcn4 is a specialized protein called a transcription factor, which helps transfer genetic information during cell growth.  The protein is activated when a cell is starving for amino acids.  What made Gcn4 interesting to the UW team was its unique mode of regulation.

“When ribosomes aren’t working at 100 percent capacity, most proteins are made less efficiently, but Gcn4 is different,” explained Dr. MacKay, a research professor of biochemistry.  “Sometimes, you actually get more Gcn4 produced even when everything else is going down.  That’s precisely what we found in the longer-lived yeast strains with mutations in the large subunit of the ribosome.”

To make the link between Gcn4 and longevity, the scientists then asked whether preventing the increase of Gcn4 would block life span extension.  In every case, cells lacking Gcn4 did not respond as strongly as Gcn4-positive cells.

“The increased production of Gcn4 in long-lived yeast strains, combined with the requirement of Gcn4 for full life-span extension, makes a compelling case for Gcn4 as an important downstream factor in this longevity pathway,” Kaeberlein said.

Although scientists don’t yet know whether Gcn4 plays a similar role in organisms other than yeast, Kennedy points out that worms, flies, mice and humans all have Gcn4-like proteins that appear to be regulated in a similar way.

“The role of TOR and translation in aging is known to be conserved across many different species, so it’s plausible that this function of Gcn4 is conserved as well,” Kennedy said.  Future research will be aimed at testing this hypothesis.

“Clearly TOR signaling is one component, and perhaps the major component, of the beneficial health effects associated with dietary restriction,” said Kaeberlein.  “The difficulty with TOR as a therapeutic target, however, is the potential for negative side effects.  As we learn more of the mechanistic details behind how TOR regulates aging, we will hopefully be able to identify even better targets for treating age-associated diseases in people.”

A team of researchers at Yale School of Medicine have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer’s recurrence and its resistance to chemotherapy.

“These results bring us closer to more effective and targeted treatment for epithelial ovarian cancer, one of the most lethal forms of cancer,” said Gil Mor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine.

Mor presented his findings recently at the annual meeting of the American Association for Cancer Research (AACR) Meeting in San Diego, California.

Cancerous tumors are made up of cells that are both cancerous and non-cancerous.  Within cancerous cells, there is a further subclass referred to as cancer stem cells, which can replicate indefinitely.

“Present chemotherapy modalities eliminate the bulk of the tumor cells, but cannot eliminate a core of these cancer stem cells that have a high capacity for renewal,” said Mor, who is also a member of the Yale Cancer Center.  “Identification of these cells, as we have done here, is the first step in the development of therapeutic modalities.”

Mor and colleagues isolated cells from 80 human samples of either peritoneal fluid or solid tumors.  The cancer stem cells that were identified were positive for traditional cancer stem cell markers including CD44 and MyD88.  These cells also showed a high capacity for repair and self-renewal.

The isolated cells formed tumors 100 percent of the time.  Within those tumors, 10 percent of the cells were positive for cancer stem cell marker CD44, while 90 percent were CD44 negative.

Mor and his team were able to isolate and clone the ovarian cancer stem cells.  They found that these cells were highly resistant to conventional chemotherapy while the non-cancer stem cells responded to treatment.  “Isolating and cloning these cells will lead to development of new treatments to target and eliminate the cancer stem cells and hopefully prevent recurrence,” said Mor.