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.

Five subtypes of muscarinic acetylcholine receptors (mAChRs) are expressed throughout the body, where they exert diverse effects, such as smooth muscle contraction, glandular secretion, thermoregulation, and regulation of behavior, learning, and cognition.  MAChRs have been implicated in schizophrenia and Alzheimer’s disease (AD), making them attractive as candidate drug targets.  Several cholinergic agonists have shown promise for treating these conditions, but most of these drugs bind to the acetylcholine binding site—which is highly conserved across receptor subtypes—and therefore have undesirable side effects.  Because of this, drug developers have recently turned to allosteric agonists, which activate receptors by binding to subtype-specific domains outside the acetylcholine binding site.  Jones et al.  Report that one such agonist, which is highly specific for M1 mAChRs, produced effects in mice similar to effects of atypical antipsychotic drugs, without producing undesirable side effects.  Moreover, the drug regulated processing of amyloid precursor protein, suggesting that it may effectively treat AD.

The differential distribution of specific proteins in axons or dendrites underlies the specialized functions of these neurites.  At least two mechanisms can create a polarized distribution of centrally produced transmembrane proteins: (1) segregation of proteins into distinct vesicles that are specifically targeted to the appropriate domain, and (2) unsorted transport followed by specific endocytosis of inappropriately expressed proteins.  This week, Bushlin et al.  Suggest that the latter mechanism can be regulated by proteins that associate with endocytic vesicles and determine their cargoes.  Knockdown of proteins involved in endocytic vesicle formation, AP180 or clathrin assembly lymphoid myeloid protein (CALM), inhibited axon or dendrite formation, respectively.  Knockdown of either protein caused VAMP2—an axonal synaptic vesicle protein that is normally endocytosed from dendrites—to be expressed in all processes, supporting a role in the establishment of polarity.  CALM knockdown also reduced surface expression of a secreted protein, suggesting that it may be involved in secretory as well as endocytic pathways.

Last week we learned that blocking expression of the AMPA receptor GluR1 subunit in motor neurons reduces dendrite growth, leading to impaired motor function.  This week, Zhou et al.  Begin to unravel the molecular mechanisms tying GluR1 to activity-dependent dendritic growth.  Intracellularly, glutamate receptors interact with membrane-associated guanylate kinases (MAGUKs)—scaffolding proteins that form the postsynaptic density and anchor signaling and other effector molecules near receptors.  GluR1 interacts with the MAGUK synapse-associated protein 97 (SAP97).  Overexpression of SAP97 increased dendritic branching, whereas SAP97 knockdown decreased branching in motor neurons.  This effect was blocked by an AMPA receptor antagonist.  Interaction between GluR1 and SAP97 was required for either to enhance dendritic branching, but only because the interaction localizes SAP97 to the plasma membrane.  If SAP97 was targeted to the membrane by the addition of a palmitoylation sequence, its effects on dendritic branching were restored in the absence of direct interaction with GluR1.

An international team of researchers led by Professors Mark Caulfield and Patricia Munroe, from the William Harvey Research Institute at Barts and The London School of Medicine and Dentistry with Chris Cheeseman at the University of Alberta in Canada and Kelle Moley at the University of Washington in USA, have shown that the SLC2A9 gene, which encodes a glucose transporter, is also a high-capacity urate transporter, and thus possibly a new drug target for gout.  Their findings are published in this week’s PloS Medicine (7 October 2008).

Several urate transporters have already been identified but recently, using an approach called genome-wide association scanning, Caulfield and others found that some genetic variants of a human gene called SLC2A9 are more common in people with high serum urate levels than in people with normal levels.  SLC2A9 encodes a glucose transporter (a protein that helps to move the sugar glucose through cell membranes) and is highly expressed in the kidney’s main urate handling site.  Professor Caulfield and his team investigated the possibility that the protein made by the SLC2A9 gene might be a urate transporter and tested whether genetic variations in SLC2A9 might be responsible for the association between serum urate levels and high blood pressure.

The team first expressed SLC2A9 in frog eggs, a type of cell that does not have its own urate transporter.  They found that SLC2A9 transported urate about 50 times faster than glucose, and that glucose facilitated SLC2A9-mediated urate transport.  Similarly, over expression of SLC2A9 in human embryonic kidney cells more than doubled their urate uptake.  Conversely, when the researchers used a technique called RNA interference to reduce the expression of mouse SLC2A9 in mouse cells that normally makes this protein, urate transport was reduced.  Researchers then looked at two genetic variations within SLC2A9 that vary between individuals (so-called single polynucleotide polymorphisms) in nearly 900 men who had had their serum urate levels and urinary urate excretion rates measured.  They found that certain genetic variations at these two sites were associated with increased serum urate levels and decreased urinary urate excretion.  Finally, the researchers used a statistical technique called meta-analysis to look for an association between one of the SLC2A9 gene variants and blood pressure.  In two separate meta-analyses that together involved more than 20,000 participants in several studies, there was no association between this gene variant and blood pressure.

Overall, these findings indicate that SLCA9 is a high capacity urate transporter, and suggest that this protein plays an important part in controlling serum urate levels.  They provide confirmation that common genetic variants in SLC2A9 affect serum urate levels to a marked degree, although they do not show exactly which genetic variant is responsible for increasing serum urate levels.  They also provide important new insights into how the kidneys normally handle urate and suggest ways in which this essential process may sometimes go wrong.  The findings could eventually lead to new treatments for gout and possibly for other diseases that are associated with increased serum urate levels.

Professor Mark Caulfield said: “This MRC funded study shows how a team of international researchers can find a completely unsuspected mechanism for urate handling in the kidney.  Such discoveries could pave the way for new medicines.”

New research suggests that the identification and examination of key cell signaling events required for initiation and progression of cancer might be best accomplished at the single cell level.  The research, published by Cell Press in the October issue of the journal Cancer Cell, provides new insight that may lead to better diagnosis and treatment of some complex cancers.

Recent advances in flow cytometry, a technique that allows detailed examination of individual cells, have enabled simultaneous measurement of cell type and signaling pathways.  Lead study authors Dr. Garry P. Nolan from the Stanford University School of Medicine and Dr. Mignon L. Loh from the UCSF Children’s Hospital and the Helen Diller Family Comprehensive Cancer Center were interested in determining whether examination of cellular signaling abnormalities caused by genetic mutations associated with cancer could provide a precise correlation between aberrant signaling events and disease physiology.

“We had a strong hunch that we could use ‘deranged’ cellular signaling to track how cancer cell populations behave at diagnosis through therapy, as well as during remission or return of the cancer,” explains Dr. Nolan.  “By measuring how signaling proteins respond to certain stimuli at diagnosis and which are modified by resistant cancers, we are essentially monitoring key highways that cancers use to drive their own growth.  The advantage of diagnosing a patient’s cancer at the single cell level provides us an approach for early detection of cancer and yield insights into how cancer cells are responding or adapting to therapy.  A byproduct of the single cell technique, when appropriately extended, is that we should eventually be able to predict those pathways cancer cells might be using to circumvent current therapies and more intelligently direct the patient towards alternative treatments.”

The researchers focused on juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative disorder of young children.  JMML is difficult to diagnose and has a complex molecular profile.  Although genetic lesions impacting Ras signaling and alterations downstream of the activated GM-CSF receptor (both linked with inappropriate cell growth and survival) have been linked with JMML, there are very few methods for identifying therapeutic agents and assessing efficacy in JMML patients.

The researchers used flow cytometry to profile signaling at the single cell level, including molecules associated with GM-CSF and Ras signaling, for the presence of primary JMML cells with altered signaling behavior that correlated with disease physiology.  Cells samples came from JMML patients, healthy individuals and patients with other myeloproliferative disorders, some who had initially been diagnosed with JMML.  An unexpected STAT5 signaling signature was seen in most of the JMML patients, suggesting a critical role for JAK-STAT signaling in the biological mechanism of this cancer and suggesting potential targets for future therapies.

“This work successfully used single-cell profiling to follow patients over time and show that disease status in JMML – at diagnosis, remission, relapse and transformation – was indicated by a subset of cells with an abnormal signaling profile,” says Dr. Loh.  “Revealing cell subpopulations, even rare cells, that are associated with disease opens additional avenues for measuring minimal residual disease, assessing biochemical effects of targeted therapies at the single cell level and understanding drug actions and mechanisms of diseases of heterogeneous origins and manifestations in diverse patient populations.”

To understand where fat comes from, you have to start with a skinny mouse.  By using such a creature, and observing the growth of fat after injections of different kinds of immature cells, scientists at the Howard Hughes Medical Institute and Rockefeller University have discovered an important fat precursor cell that may in time explain how changes in the numbers of fat cells might increase and lead to obesity.  The finding, published online in this week’s issue of the journal Cell, could also have implications for understanding how fat cells affect conditions such as diabetes and cardiovascular disease.

“The identification of white adipocyte progenitor cells provides a means for identifying factors that regulate the proliferation and differentiation of fat cells,” says senior author Jeffrey Friedman, who is the Marilyn M. Simpson Professor at Rockefeller and a Howard Hughes Medical Institute investigator.

Obesity, a major public health problem in the United States and increasingly in much of the Western world, results, in part, from an increase in the mass and number of white fat cells.  Because white fat cells are post-mitotic, meaning that they cannot divide, scientists have hypothesized that a population of fat precursor cells must exist in the fat depot in order to produce new fat cells.  But identifying these fat precursor cells has been difficult.

With the assistance of researchers in Rockefeller’s Flow Cytometry Resource Center, first author Matt Rodeheffer, a postdoctoral associate in Friedman’s Laboratory of Molecular Genetics, used a cell sorting technique called fluorescence-activated cell sorting, or FACS, to search for cell populations that could produce fat in cell cultures and identified two such populations.

To determine if these cells could develop into fat cells in living animals, Rodeheffer injected these cell populations into the fat depots of a genetically engineered mouse, developed at NIH, called fatless, which lacks white fat and mimics a condition in humans called lipodystrophy that also results in diabetes.

Rodeheffer found that only one of the isolated cell populations, which express the CD24 cell-surface marker protein, produced fat tissue in the fatless mouse.  This population normally represents only .08 percent of the non-adipocyte population in adipose tissue.

An imaging assay recently developed by co-author Kivanç Birsoy, a graduate student in Friedman’s laboratory, enabled Rodeheffer to observe the CD24-expressing cells form fat in a living animal.  Birsoy’s technique uses another animal strain called the leptin-luciferase mouse, in which the visibly detectable marker luciferase is expressed under the control of the promoter of the gene that produces the hormone leptin.  In this mouse strain the luciferase marker gene only switches on in mature fat cells, and provides a non-invasive way of watching immature fat cell precursors develop into mature fat cells in a living animal over time.

“I injected the CD24+ cells which represent a very small population of cells in normal adipose tissue into a site where the fat would normally develop in the fatless mouse, and I found that a normal sized fat depot forms at the site of injection,” says Rodeheffer.

Rodeheffer also found that the injection of the fat-producing cells corrects the fatless mouse’s diabetes, and the fat cells secrete adipocyte-specific signaling proteins called cytokines.  Both of these results confirm that the cells produced in the fatless mouse are functional fat cells.

“This finding gives us a better understanding of the basic biology of adipose tissue and opens the door for us and for other researchers to be able to study these cells in living animals and determine the molecular factors that regulate formation of adipose tissue,” says Rodeheffer.  “We then can potentially study how the growth and differentiation of these cells are regulated in obesity and determine whether or not the molecular events that are involved in the regulation of adipose tissue are contributing factors to other pathologies, such as diabetes and cardiovascular disease, that are associated with obesity and metabolic syndrome.”

A bacteria cell’s ‘crisis command centre’ has been observed for the first time swinging into action to protect the cell from external stress and danger, according to new research out today (3 October) in Science.

The research team behind today’s study says that finding out exactly how bacteria respond and adapt to stresses and dangers is important because it will further their understanding of the basic survival mechanisms of some of the most resilient, hardy organisms on Earth.

The crisis command centre in certain bacteria cells is a large molecule, dubbed a ’stressosome’ by the scientists behind today’s research.  These cells have around 20 stressosomes floating around inside them, and although scientists knew they played an important role in the cell’s response to stressful situations, the complexities of this process had not been fully understood until now.

If a bacteria cell finds itself in a dangerous situation for example, if the temperature or saltiness of the bacteria’s environment reach dangerous levels which threaten the survival of the bacteria -a warning signal from the cell’s surface is transmitted into the cell.

Using cutting edge electron microscopy imaging techniques the authors of the new research observed that the stressosomes receive this warning signal, and in response several proteins called RSBT break away from the large stressosome.  This breakaway triggers a cascade of signals within the cell which results in over 150 proteins being produced proteins which enable the cell to adapt, react and survive in its new environment.

Professor Marin van Heel from Imperial College London’s Department of Life Sciences, one of the corresponding authors of the study, explains: “The cascade of events inside bacteria cells that occurs as a result of stressosomes receiving warning signals leads to particular genes inside the cell being transcribed more.  This means that some genes already active inside the cell are ‘turned up’ so that levels of particular proteins in the cell increase.  These changes to the protein make-up of the cell enable it to survive in a hostile or challenging environment.”

Dr Jon Marles-Wright from Newcastle University says: “Our work shows that cells respond to signals much like a dimmer on a light switch.  Now we’ll be building on this to work out how nature controls that dimmer switch.  We wouldn’t have been able to carry out this work without access to the Diamond synchrotron Light Source which has enabled us to examine the structures of individual stressosome proteins at atomic resolution.”

Dr Tim Grant, one of Imperial’s post doctoral researchers, adds that the key to bacteria cells’ success at surviving in rapidly changing environments is their speedy response: “The cell’s stressosomes are very good at their job as crisis command centres because they provide a very fast effective response to danger.  The chain reaction they kickstart produces results really quickly which enables bacteria to adapt to changes in their surroundings almost instantaneously.”

The team is now planning to collect very high resolution data of the stressosome complex on the world’s newest high-resolution cryo electron microscope, the FEI “KRIOS” that has just been installed in the Max Planck Institute in Martinsried, Germany.  Improving the resolution of the stressosome structure by a factor of two will lead to a resolution range normally only attainable by X-ray crystallography and will allow the researchers to directly see the amino-acid components of this fascinating complex.

Reported in the Oct. 3, 2008 issue of Cell, the findings–from a study in mice–point to a completely new approach to treating and preventing obesity in humans.  The discovery also offers hope for new ways to treat related disorders, such as type 2 diabetes and cardiovascular diseases–the most prevalent health problems in the United States and the rest of the developed world.

Led by Dongsheng Cai, an assistant professor of physiology at the UW School of Medicine and Public Health, the researchers looked specifically at the hypothalamus–the brain structure responsible for maintaining a steady state in the body–and for the first time found that a cell-signaling pathway primarily associated with inflammation also influences the regulation of food intake.  Stimulating the pathway led the animals to increase their energy consumption, while suppressing it helped them maintain normal food intake and body weight.

The research stems from recent explorations into the problem called metabolic inflammation, a by-product of too much food or energy consumption.  Unlike the classical inflammation typically observed in infections, injuries and diseases such as cancer, the metabolic inflammation seen in obesity-related diseases is much milder, doesn’t lead to overt symptoms or cause tissues damage.

“Metabolic inflammation is a chronic, low-grade condition consisting of inflammatory-like responses at the molecular level.  It has many downstream consequences,” says Cai.  “It causes cellular dysfunction, which can decrease the regulation of several physiological processes, including metabolism.”

Scientists believe that metabolic inflammation may be at the core of many chronic, obesity-related metabolic disorders that are so common today, he adds.

Cai and his team zeroed in on NF-kappaB, a protein complex that can be activated specifically by IKKbeta to induce inflammatory reactions in many cell systems.

In earlier studies at Harvard, Cai and colleagues found that the pathway interrupted sugar, fat or protein metabolism in tissues where metabolism typically takes place–liver, fat and skeletal muscle.  Feeding mice high-sugar and high-fat diets activated the pathway in these tissues.

Once he arrived at the SMPH three years ago, Cai began to consider whether metabolic inflammation might affect “higher-up” players in the central nervous system, particularly the hypothalamus.  This brain structure is a critical master regulator of appetite and energy balance, and also controls metabolism in the peripheral tissues he had studied before.  But nobody knew how the hypothalamus might contribute to the development of metabolic diseases such as obesity and diabetes.

“We wanted to learn whether the pathway or pathways underlying metabolic inflamm ation could affect metabolism regulators in the central nervous system,” he says.

In the current study, Cai and his team found first that IKKbeta/NF-kappaB does indeed exist in specific neurons in the hypothalamus.  The pathway is much more abundant in the hypothalamus than in peripheral tissue, and it normally remains inactive in the brain.

The researchers next showed that over-nutrition through high-fat diet feeding activates IKKbeta/NF-kappaB, specifically in neurons in the hypothalamus.

“When we knocked out the IKKbeta gene to suppress NF-kappaB activity in these neurons, the animals were significantly protected from energy over-consumption and obesity development,” Cai says.

The researchers also examined a cell component called the endoplasmic reticulum (ER), shown recently to be involved in metabolic diseases involving over-nutrition, to see if it might play a role in linking over-nutrition to activate IKKbeta/NF-kappaB in the hypothalamus.

“At the intracellular level, when the ER is challenged with over-nutrition, this leads to ER stress, which can push the IKKbeta/NF-kappaB pathway to an active state, although the involved reactions could be quite complicated,” Cai says.

In several experiments, the researchers found that ER stress caused by over-nutrition activated IKKbeta/NF-kappaB in the hypothalamus.  Suppressing ER stress in the central nervous system significantly preserved normal regulation of food intake and prevented obesity.

Cai says there’s still a lot of work to be done.  His group has begun studying IKKbeta/NF-kappaB’s connections to other pathways and regulations in the hypothalamus.

“The ultimate goal will certainly be to identify a selective and effective suppressor of the pathway to target related neurons,” he says.

But Cai continues to look at the big picture, seeking answers to questions such as: “How does the environment connect to the genetics that seem to underlie the obesity epidemic?  What are the key steps that have led to the dramatic rise of diabetes in the past three decades?  And Why can’t the body adjust to changes that have occurred in the way people eat and what they eat?”

Researchers may have found the key to engineering plants capable of thriving in environments laden with toxic aluminum, according to a report published online on October 2nd in Current Biology, a Cell Press publication.  Aluminum (Al)—a metal that is generally plentiful in the earth’s crust—causes particular problems for farmers in South America, Africa, and Indonesia, where acidic environments turn the metal into a form that stunts the growth of plants and especially plant roots.

” We found that a single change in one plant factor required for monitoring of and response to DNA damage results in a profound increase in Al tolerance,” said Paul Larsen of the University of California-Riverside.

That discovery was unexpected, he said, because scientists had believed Al could have a wide range of detrimental effects, binding to virtually any negatively charged molecule within cells.  If that were true, getting around Al toxicity would be no easy task since any single change in plants would result in only incremental increases in Al tolerance.

” Surprisingly, we found that elimination of just one factor results in a mutant root that can now thrive in an Al toxic environment,” Larsen said.  The critical factor, known as AtATR, serves as a “checkpoint” for cell division, he explained.  Its job is to assess whether a cell should divide or not, on the basis of the integrity of the cell’s DNA.  “Mutations that disrupt the function of AtATR effectively destroy this self-assessment activity and allow cells that otherwise would be forced to differentiate [into mature plant tissue] to continue dividing.”

The results present a new view of the causes of Al toxicity.  Rather than suffering from the metal’s cumulative toxic effects as had been believed, it appears Al itself triggers the AtATR-controlled self-assessment pathway to shut down growth.

The findings made in the model plant Arabidopsis offer “readymade” tools for genetically engineering crop plants incapable of restricting root growth in response to Al toxicity, Larsen said.  He anticipates that introduction of the mutant versions of AtATR into crop plants would override the existing assessment mechanisms and allow for continued cell division in soils that would normally inhibit root growth.

The new results may offer insight into Al toxicity not only in economically important agricultural crops, but also in animals, given that ATR genes are universally found in plants and animals, where they serve in various capacities related to DNA-damage assessment.

” To date, no one has been able to discern which targets of Al are critical to the manifestation of Al toxicity in either plant or animals, partly due to the predicted complexity of Al toxicity,” he said.  “This work clearly argues that DNA damage and response to this damage is paramount.”

Breakthroughs in cytogenetic technologies, which focus on subtle alterations in genes and chromosomes, are enabling a new level of detail and accuracy in the diagnosis of complex and unexplained developmental problems in children.

The availability of this new information can help clinicians shift to a “genotype first” model of diagnosis, according to David H. Ledbetter, PhD, Woodruff professor of human genetics at Emory University and director of the Division of Medical Genetics.

Ledbetter’s editorial on “Cytogenetic Technology–Genotype and Phenotype,” is published online this week by the New England Journal of Medicine.  It accompanies an article by Heather Mefford and colleagues about using new cytogenetic technologies to identify microdeletions and microduplications in a specific region of chomosome 1q21.1 in patients with unexplained mental retardation, autism or congenital anomalies

Cytogenetic arrays that reveal DNA microdeletions and additions, including single-copy changes of a few hundred base pairs, beadchips that detect single-nucleotide polymorphisms (SNPs) and tests called comparative genomic hybridization have led to an exciting renaissance of genetics-based syndrome delineation, says Ledbetter in his editorial.

“In the early 1960s we began discovering the relationship between chromosome imbalance and diseases and syndromes, such as Down syndrome,” says Ledbetter.  “This was based on identifying multiple patients with the same cytogenetic abnormality and similar clinical symptoms.  Ever since then, technology breakthroughs have allowed us to identify new syndromes and ever more subtle genetic differences.”

The current proliferation of new genetic information has led researchers to discover that many small genetic variations are common and mostly benign in the human population.  This means the relationship between DNA variations and disease must be analyzed even more carefully in order to find accurate connections.  In order to prove that a genetic difference is directly related to a particular syndrome, notes Ledbetter, researchers must show that the difference is never found in normal control individuals or at least is found with significantly less frequency.

Also, researchers have found that a particular genetic variation may have only a mild effect in a parent but a much more severe effect in a child who inherits the same variant.  And a group of children may have a variety of different problems resulting from the same gene variation.  Whole-genome cytogenetic arrays are becoming much more common, however, which is bringing genetic testing to the level of everyday medicine.

“So many variations of developmental disorders and syndromes have been discovered that genetic testing has become essential for making a specific clinical diagnosis,” says Ledbetter.  “Although more information has made the job of a diagnostician even more challenging, it also is leading to more accurate diagnoses and should lead to much more effective treatments.”

Some of the most challenging obstacles limiting the reprogramming of mature human cells into stem cells may not seem quite as daunting in the near future.  Two independent research papers, published by Cell Press in the September 11th issue of the journal Cell Stem Cell, describe new tools that provide invaluable platforms for elucidating the molecular, genetic, and biochemical mechanisms associated with reprogramming.  The new findings also offer considerable hope toward making the reprogramming process more therapeutically relevant.

Although scientists have successfully reprogrammed mature human skin cells into induced pluripotent stem (iPS) cells by expressing a few key transcription factors, the conversion has been extremely inefficient.  “Little is known about the mechanisms by which reprogramming occurs, in part because of the low efficiency,” says senior study author Dr. Konrad Hochedlinger from the Harvard Stem Cell Institute.  In addition, the iPS cells created thus far have been generated with retroviruses and noninducible lentiviruses, both of which have major limitations that are not compatible with clinical applications.

The Hochedlinger group created a drug-inducible viral system to generate human iPS cells that were molecularly and functionally similar to human embryonic stem cells.  This method was unique in that it allowed the researchers to create iPS cells by using the drug doxycycline to control expression of the necessary factors that had been delivered to the cells with viruses.

The researchers then found that when doxycycline was removed and these “primary” iPS cells differentiated to mature cells, another exposure to the drug reactivated the genes required for reprogramming and induced generation of “secondary” iPS cells at a frequency that was far greater that the initial “primary” conversion.  The idea of generating these secondary cells was conceived in previous experiments with mice performed in the lab of Dr. Rudolf Jaenisch from the Massachusetts Institute of Technology.

“The secondary system will enable chemical and genetic screening efforts to identify key molecular constituents of reprogramming, as well as important obstacles in this process, and will ultimately lend itself as a powerful tool in the development and optimization methods to produce human iPS cells,” explains Dr. Hochedlinger.

In a separate paper, Dr. Jaenisch’s group reports on their success in deriving human secondary iPS cells using doxycycline-inducible transgenes.  “The drug-inducible system we describe represents a novel, predictable, and highly reproducible platform to study the kinetics of iPS cell generation,” says Dr. Jaenisch.  “Further, the genetic homogeneity of secondary cells makes chemical and genetic screening approaches to enhance reprogramming efficiency or to replace any of the original reprogramming factors feasible.”

Both research teams found that generation of secondary human iPS cells required less time than the initial reprogramming.  Interestingly, the time required to generate iPS cells varied among the types of skin cells that were used.  For instance, human fibroblasts required several weeks, while keratinocytes required only about 10 days.  “The fast kinetics of reprogramming observed for keratinocytes suggests that these cells would be useful for development and optimization of methods to reprogram cells by transient delivery of factors,” suggests Dr. Hochedlinger.

The combined results from both research groups represent a major advance toward more efficient strategies for reprogramming differentiated human cells into iPS cells.  The methods described here will not only provide critical insight into the reprogramming process, but also, because of the abbreviated time frame, may lead to the generation of cells that will be amenable for therapies, as reprogramming might be achievable without the prohibitive viruses or genetic modifications.

Scientists investigating the mechanisms of Down Syndrome (DS) have revealed the earliest developmental changes in embryonic stem cells caused by an extra copy of human chromosome 21 – the aberrant inheritance of which results in the condition. Their study is published online today (Thursday 4 September) in the American Journal of Human Genetics.

Lead by Dean Nizetic, Professor of Cellular and Molecular Biology at Barts and The London School of Medicine and Dentistry, the team utilised embryonic stem cells from a previously genetically engineered species of mice carrying a copy of human chromosome 21. They discovered that extra chromosome 21 - a genetic state known as trisomy 21 - disturbs a key regulating gene called NRSF or REST, which in turn disturbs the cascade of other genes that control normal development at the embryonic stem cell stage. Furthermore, they identified one gene (DYRK1A) on human chromosome 21, whose overdose in trisomy (DS) is responsible for the observed effects.

Down Syndrome belongs to the group of conditions called ‘aneuploidies’, defined by an abnormal loss or gain of genetic material, i.e. fragments of chromosomes or whole chromosomes. Aneuploidies cause congenital anomalies that are a prime cause of infant death in Europe and the USA, and are currently on the increase with advancing maternal age in European countries. The number of people with DS in Europe exceeds half a million. The condition is more common than muscular dystrophy and cystic fibrosis, but the development of new therapeutic concepts is hindered by the fact that unlike muscular dystrophy and cystic fibrosis, where a single mutated gene causing the disease is known, the entire human chromosome 21 (containing around 300 genes) still has to be dissected into individual gene-dose contributions to the DS symptoms.

Professor Nizetic, calling for further research into the components of the disturbed cascade he and his team have revealed said; “We hope that further research might lead to clues for the design of new therapeutic approaches tackling developmental delay, mental retardation, ageing and regeneration of brain cells, and Alzheimer’s disease. In other words, we hope our work will open new routes to tackle the genetics of these health disorders, approaching them from the “back entrance”, as dominant component-symptoms of Down Syndrome.”

The studies results may explain why kids of older dads are more likely to have some genetic disorders, and why those disorders are more common than expected…

In a cruel irony, testis cells carrying the mutation that causes Apert’s syndrome are fitter than normal cells, even though children born from sperm derived from those cells are weakened by fused fingers, toes and skulls, a new study has found.

The research, to be published in the Proceedings of the National Academy of Sciences Online Early Edition during the week of July 14-18, can explain why the syndrome is unexpectedly common, and why sperm from older men carry the mutation more frequently than expected.

The likelihood that a child from an older father inherits this and similar genetic diseases is approximately 10- to 20-fold greater than that of a younger father, yet the molecular reasons behind it have been elusive, said USC biologist Norman Arnheim, who co-led the study with USC’s Peter Calabrese.

Calabrese, Arnheim and two other USC colleagues found the strongest evidence yet that testis cells carrying the mutant gene causing Apert’s syndrome have a survival advantage over non-mutant cells. This means that as a man ages, the number of mutant cells rises exponentially, as does the sperm descended from them.

Because so much DNA is constantly being copied, small errors often occur. Apert’s syndrome is caused by one of two simple switches on a gene located in a man’s sperm.

But geneticists have puzzled over why Apert’s syndrome occurs 100 to 1000 times more often than would be expected from random, spontaneous copy errors.

Thanks to a method developed by Arnheim’s lab that divides the testis into about 200 units, the scientists observed that cells with mutated DNA are clustered in specific areas, rather than distributed evenly, as would be expected if the copy errors simply occurred more frequently.

While the researchers have seen this before, this study is the first to test both Apert’s syndrome mutations in testes from both young and old individuals in this way.

Comparing computer models with observed data, the scientists were able to demonstrate that the high frequency of the disease is not due to an increased chance of a mistake being made when the gene is copied, as has been widely proposed in the past.

Instead, the concentrated areas observed in the testes could be explained by a selective advantage of the mutant cells over non-mutant ones, meaning that mutant lineages would grow in number over time, thus increasing the chances that more sperm will contain mutant genetic material.

This seems counter-intuitive, since when we think of natural selection, we often think of beneficial traits, like a mutant butterfly with camouflaged wings, which escapes predators and passes this advantageous color to its offspring.

But in the case of Apert’s syndrome, the gene switches end up making the mutant testis cell fitter, while this is not the case in the humans who develop from the resulting sperm.

“It just seems so odd that the testis that causes such a harmful disease for the kid apparently has an advantage over cells without the mutation,” Calabrese said. While theories have been suggested, it’s not yet known what this advantage is for sure, he added.

This evolutionary explanation, which has been proposed but rarely tested, may hold true for other genetic disorders such as achondroplasia, the most common form of dwarfism, as that condition is also linked to a single gene substitution.

“I think it raises the possibility that there might be a larger class of genetic diseases that are the result of a selective advantage when the mutation occurs,” Arnheim said.

If scientists are able to pin down the molecular mechanism that enables this advantage, there could in theory be ways to counteract it, although such thinking is highly speculative, he added.

The study is also of interest since some mutations in the same genes involved in Apert’s syndrome and achondroplasia (FGFR2 and FGFR3) appear to be involved in some types of cancer. While little is known about the mechanisms behind those mutations, such information might eventually help explain the molecular basis for the advantage in the testis.

The paper’s other authors were Soo-Kyung Choi and Song-Ro Yoon. The research was funded in part by grants from the National Institute of General Medical Sciences and the Ellison Medical Research Foundation.

New data, generated by David Ornitz and colleagues, at Washington University School of Medicine, St. Louis, have indicated a crucial role for signaling pathways that involve the protein sonic hedgehog in maintaining the blood vessels that supply the mouse heart and keep it beating.  These data have implications for drug development as they suggest that antagonists of hedgehog signaling pathways, such as those being developed as anticancer therapeutics, might have unwanted side effects.

In the study, mice lacking the ability to mediate hedgehog signaling in cells that form part of the blood vessels that supply the heart were found to die of heart failure.  This was because in the absence of hedgehog signaling the blood vessels of the heart were lost, meaning that the heart cells were no longer supplied with enough oxygen and died.  Although these data indicate a need for caution when developing clinical antagonists of hedgehog signaling, it is possible that the degree of inhibition needed to have a clinical effect on tumor development might not have the effect on blood vessels of the heart that completely eliminating expression of the protein does.