August 9, 2013
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Cognitive decline, memory impairment, and anxiety are all typical parts of normal aging. New research published in the Journal of Neuroscience from the lab of Dr. Remi Quirion at McGill University sheds light on cellular mechanisms in the brain that may underlie this decline. Dynorphins, endogenous opioid-like proteins, appear to be increased in aged brains, and through activation of specific opioid receptors can decrease the function of synaptic connections releasing the amino acid neurotransmitter glutamate. Glutamate is the most common neurotransmitter in the brain and acts on several different receptor types, but the effect of dynorphin appears to arise through reduction in activity of a specific group of glutamate receptors, the metabotropic glutamate receptors (mGluRs). mGluRs can be important for different forms of synaptic plasticity, or the remodeling of neural connections, and appear to be important for decreasing the strength of certain synapses in order to properly shape neural circuits that underlie learning and memory formation. The hypothesis that Dr. Quirion’s group pursued was that increased dynorphin expression in aging brains leads to decreased mGluR activation which in turn limits the synaptic plasticity required for memory and other cognitive processes. To examine this, the group compared anatomical, physiological, and behavioral characteristics of wild-type mice and mice that had the dynorphin precursor, prodynorphin, knocked out. In general, young and middle-aged wild-type mice did not differ from young and middle-aged prodynorphin knockout mice. However, many differences were observed between old wild-type mice and old prodynorphin knockout mice. Old prodynorphin knockout mice had higher mGluR expression than old wild-type mice, and they exhibited mGluR-mediated plasticity at a level similar to young and middle-aged mice. Additionally, on behavioral tests of memory, learning, and anxiety, the prodynorphin knockout mice performed better than and displayed less anxiety behaviors than old wild-type mice. In fact, old prodynorphin knockout mice performed similarly to young and middle-aged mice on the behavioral tasks. To confirm that these changes were mediated by mGluRs and dynorphin receptors, the authors also performed behavioral tests in mice that had received pharmacological treatment. In old wild-type mice, pharmacologically increasing the activity of mGluRs or blocking the activity of dynorphin receptors resulted in greatly improved performance on the memory and learning tasks and in decreased anxiety behaviors. Conversely, in old prodynorphin knockout mice pharmacologically inhibiting the activity of mGluRs resulted in poorer performance on memory tasks and in increased anxiety behaviors. Together, these data clearly demonstrate that increases in dynorphin during normal mammalian aging can cause a reduction in mGluR-mediated synaptic changes that underlie learning, memory, and anxiety, and they identify a pathway that can be targeted to improve cognitive ability in the aging population.
July 5, 2013
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A few weeks ago I reviewed a paper that demonstrated that pluripotent stem cells could be induced by repressing muscleblind-like RNA binding (MBNL) proteins in differentiated cells. Continuing in this vein, new research published in Nature Cell Biology from the lab of Dr. Connie Eaves at the BC Cancer Agency identifies a pathway crucial for the maintenance of the self-renewal properties of hematopoietic stem cells (HSCs). Using array analysis and quantitative polymerase chain reaction, the authors found that the Lin28b gene is expressed much more highly in mouse fetal liver HSCs than in adult bone marrow HSCs, which have greatly reduced self-renewal capabilities compared to fetal liver HSCs. Adult bone marrow HSCs transfected with the Lin28 gene and subsequently transplanted in to mice of at least 10 weeks of age have increased self-renewal capabilities and also show decreased levels of let-7 microRNA, the production of which is inhibited by Lin28b. Additionally, flow cytometry experiments demonstrated that Hmga2, a protein that is known to be inhibited by let-7, was increased in adult bone marrow HSCs following Lin28 transfection. Directly increasing Hmga2 levels in adult bone marrow HSCs increased the self-renewing capabilities of these cells, whereas fetal liver cells lacking Hmga2 have greatly reduced self-renewing capability. Overall, this study demonstrates that the self-renewing capability of mouse hematopoietic stem cells is regulated by the Lin28 – let-7 – Hmga2 axis, which is down-regulated within a few weeks following birth. Thus, this pathway offers a potential target for the development of tissue-specific self-replicating cells, which promise to be very important for the treatment of a variety of diseases.
June 21, 2013
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Arthritis is a highly prevalent disorder, affecting about 1 in 5 people in North America, and is characterized by joint inflammation, which results in pain and reduction in joint mobility. Underlying these symptoms is the release of a peptide called substance P, so called because of its role in signalling pain. New research published in the Journal of Neuroscience from the lab of Dr. Alfredo Ribeiro-da-Silva at McGill University indicates that joint pain associated with arthritis may be due in part to increased activation of substance P-releasing nerve cells caused by inappropriate sprouting of sympathetic nerve fibers. The authors induced arthritis, which was characterized by joint edema and increased pain responses, in the hind ankle joint of rats. Using immunohistochemistry, the authors found that sympathetic nerve fibers, which normally innervate blood vessels in the skin to regulate blood-flow, sprouted to innervate the synovial membrane surrounding the ankle joint and the upper dermis covering the ankle joint four weeks after the arthritic phenotype was induced. Interestingly, these newly sprouted sympathetic nerve fibers closely associated with peptidergic nerve cells, which release substance P and are associated with transmitting pain responses. This result suggests that sympathetic nerve sprouting can increase the activity of substance P-containing neurons, exaggerating pain responses and inflammation. Consistent with this, pharmacologically blocking sympathetic nerve responses in these rats shifted the pain threshold back near normal. The authors also found that the expression of mature nerve growth factor (NGF), which is necessary for sympathetic neuron growth and survival, was increased near the ankle joint in which arthritis had been induced. The authors suggest that activity of peptidergic nerve cells increases joint and skin inflammation which initiates an immune response leading to increased production of mature NGF. The increase in NGF leads to sympathetic nerve sprouting, a further increase in peptidergic nerve cell activity, and ultimately to hyperactive pain signaling. These findings suggest that decreasing the production of mature NGF near arthritic sites can decrease sympathetic nerve sprouting and reduce arthritis associated pain. Additionally, localized inhibition of sympathetic neurotransmission may be a way to provide short-term arthritis pain relief.
June 14, 2013
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Embryonic stem cells are pluripotent, meaning they have the ability to differentiate into multiple cell types. Due to this, embryonic stem cells have the potential to be used in cell-based therapies to treat diseases in which specific cell types are lost, such as Alzheimer’s or diabetes, or to promote recovery following traumatic events, such as spinal cord injury or stroke. However, the use of embryonic stem cells is controversial for a number of ethical reasons, with specific concern surrounding how they are harvested. Induced pluripotent stem cells (iPSCs) offer an alternative to embryonic stem cells, because they can be derived from differentiated host tissue. Because changes in gene expression within embryonic stem cells lead to their differentiation and loss of pluripotency, iPSCs can be produced from differentiated cells by essentially reversing these changes. New work published in Nature from the lab of Dr. Benjamin Blencowe at the University of Toronto identifies muscleblind-like RNA binding proteins (MBNL1 and MBNL2) as regulators of specific gene splicing events that differ between embryonic stem cells and differentiated cells. Using high-throughput sequence profiling and quantitative polymerase chain reaction, the authors found that MBNL proteins are expressed less in embryonic stem cells and iPSCs than in differentiated cells. This led the authors to hypothesize that MBNL proteins repress expression of certain sequences of RNA that maintain the pluripotent state of embryonic stem cells. To examine if this was the case, the authors used small interfering RNA to decrease the amount of MBNL1 and 2 protein expressed in cultured mouse and human cells. In approximately half of these cells, decreasing the MBNL proteins altered the splicing of the FOXP1 gene, a gene important for triggering a switch between embryonic stem cells and differentiated cells, and returned the cells to an embryonic stem cell-like pattern of FOXP1 expression. It was also possible to do the opposite: over-expression of the MBNL1 and 2 proteins in mouse embryonic stem cells caused these cells to quickly adopt the FOXP1 splicing pattern seen in differentiated cells. Importantly, knockdown of MBNL proteins increased the level of several transcription factors that are critical for maintaining pluripotency of embryonic stem cells, and significantly increased the number of iPSC colonies. These results demonstrate that MBNL proteins 1 and 2 are directly involved in the control of embryonic stem cell pluripotency, and that reduction of their expression in differentiated cells can lead to induction of iPSCs. MBNL expression is therefore an attractive therapeutic target to create pluripotent cells for use in cell-based therapies, as the use of iPSCs eliminates many of the ethical concerns surrounding the use of embryonic stem cells for such therapies.
May 31, 2013
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During development several proteins guide the mapping of blood vessels throughout our body, providing different cues to direct them where they should go and, equally as important, not go. New research published in Nature from the lab of Dr. Sabine Cordes at Mt. Sinai Hospital in Toronto describes the role of a protein called gumby in the formation of the microvasculature. Mice lacking gumby displayed normal patterning of the major vasculature, but patterning of the smaller vascular networks in the head and trunk of the mice was greatly disrupted. Genetic mapping allowed the authors to determine that mutations in the Fam105b gene were underlying the defects observed in the “gumby mice”, and identified the gumby protein as a deubiquitinase. This means that gumby can counteract ubiquitination, a process in which proteins are tagged for a certain fate; this fate may be degradation, movement within a cell, or initiation of cellular processes. The authors also found that gumby interacts with a ubiquitinating complex called LUBAC, and that together these proteins can modulate the Wnt pathway, a pathway important in the development of the vasculature. Because LUBAC and gumby serve opposing functions, the interaction of these two proteins effectively creates a signaling axis that allows for flexibility in the management of the Wnt pathway, and ultimately the development of the vasculature. Identification of these pathways and the genes underlying them creates new possible targets for the management of disorders of vasculature mapping, as well as disorders in other systems that require significant mapping, such as the nervous system.
May 17, 2013
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Parkinson’s disease, a neurodegenerative disease characterized by motor and cognitive deficits, may be caused by mutations in the Parkin gene. The Parkin gene transcribes the Parkin protein, an enzyme which has been implicated in cellular processes including autophagy, or “cell housekeeping,” and cell survival. Recent work from the Montreal Neurological Institute and McGill University’s Department of Biochemistry published in Science magazine demonstrates the crystal structure of the Parkin protein. The crystal structure of a protein offers an excellent idea of the natural conformation a protein adopts; determining the structure of Parkin protein, surely an immense amount of work, will allow for advanced studies of how the protein normally functions, and will increase understanding of how mutations in the Parkin protein lead to the deficits seen in Parkinson’s disease.
Parkin protein normally has low basal activity. Following determination of the structure of Parkin, the authors found that it can maintain this low baseline by inhibiting itself. Additionally, by testing a number of mutations in the Parkin protein, they found that most mutations greatly reduce or abolish its already low basal activity. However, mutations directed at eliminating auto-inhibition of Parkin were able to increase activity of the protein, indicating that it is possible to bidirectionally change its activity. These experiments demonstrate the utility of knowing the crystal structure of the Parkin protein, as future studies will be able to better evaluate how the activity of the protein can be managed, which may prove very important in the treatment of Parkinson’s disease.
May 10, 2013
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Stroke is the leading cause of disability in North America, but no good treatment exists for stroke beyond a few hours of its occurrence. The damaging effects of stroke occur because nerve cells in the brain require oxygen to survive; once blood-flow to the brain is disrupted and oxygen delivery to nerve cells stops, the cells enter a state called excitotoxicity and begin to die. The best way to improve stroke outcome is to limit the amount of nerve cell death that occurs. Much pharmacological treatment has been directed at inhibiting a specific neurotransmitter receptor central to excitotoxicity, but this approach can have broad effects within the brain. New research from Dr. Yu Tian Wang’s lab at the University of British Columbia published in the Journal of Neuroscience offers a potential new target to limit nerve cell death following stroke. The researchers found that PTEN, a protein that promotes cell death once it enters the nucleus of a cell, becomes targeted to nerve cell nuclei after excitotoxicity starts; additionally, they found that a specific portion of PTEN is critical for its entry in to the nucleus. In nerve cells that were pharmacologically treated to become excitotoxic, ones that had this portion of PTEN mutated were less likely to die. Furthermore, mice injected with a peptide that inhibits the entry of PTEN in to the cell nucleus experienced less extensive physical brain damage, increased nerve cell protection, and more rapid and complete motor skill recovery following an induced stroke; these effects were seen if the peptide was delivered to mice up to 6 hours after the stroke was induced. These results indicate that limiting nerve cell death through inhibition of a downstream protein involved in excitotoxicity may be a viable new approach for stroke treatment, one which may also extend the treatment window following the occurrence of a stroke.
The use of oncolytic viruses is becoming an increasingly attractive avenue for the treatment of cancer, because these viruses are able to destroy tumor cells and also generate immune-responses directed at those same tumor cells. Using human viruses for this type of treatment may be inefficacious, because immunity may exist or quickly develop toward the virus that is being used. The use of animal viruses similar to human viruses may prove to be an effective way to avoid these potential immunity problems.
A proportion of cells within a tumor are cancer stem cells, which are often enriched in what is called a “side population” of tumor cells. These cells can self-renew, produce new cells derived from multiple cell lineages, and may increase the growth rate of tumors. Previous research from Dr. Karen Mossman’s lab at the McMaster University Immunology Research Centre demonstrated that bovine herpesvirus type 1 targets transformed human cells but not normal human cells, and new research from her lab published in Cancer Gene Therapy now demonstrates that this virus can infect and kill cancer stem cells within the tumor side population.
The researchers found that exposing a number of different breast cancer cell lines to the bovine herpesvirus 1 led to a decrease in tumor cell viability and an increase in tumor cell death. The virus also killed human breast cancer stem cells, and limited the self-renewal and cellular differentiation capabilities of these cells. Additionally, mice injected with breast cancer stem cells that had been exposed to bovine herpesvirus 1 formed much smaller tumors than mice that were injected with breast cancer stem cells that were untreated. These results offer hope that bovine herpesvirus 1 or similar viruses may be particularly useful in treating cancers, because, in addition to not targeting normal cells, they are effective in killing the particularly harmful cancer stem cell population, and their use could also limit immunity issues that may reduce treatment efficacy.
April 26, 2013
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Inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis, are autoimmune disorders in which persistent bowel inflammation leads to physical damage of the intestinal tract. Research out of the Inflammation Research Network at the University of Calgary, and published in the Proceedings of the National Academy of Sciences, offers a cool novel target for the treatment of IBD. Just like we have receptors that signal heat, evident when we get a hot or burning sensation when eating a chili pepper, we also have receptors that signal cold, which can be activated by compounds such as menthol. The researchers found that a receptor that signals cold (TRPM8) is expressed more highly in colon samples from humans with Crohn’s disease than in samples from those that do not have IBD. This finding suggests that the TRPM8 receptor is up-regulated in inflamed tissue in an attempt to cool it, similar to how controlled cooling is used to treat sites of traumatic injury.
To investigate the potential of the TRPM8 receptor as a target for inflammation reduction, the researchers performed experiments in mice in which colitis had been induced. Mice with induced colitis expressed the TRPM8 receptor more highly in their colon than mice that did not have induced colitis. The mice with induced colitis also had high levels of cytokines, signalling molecules that can promote inflammation, in their colon; activating the TRPM8 receptor with a molecule called icilin reduced these cytokine levels to normal. Activation of the TRPM8 receptor in mice with induced colitis also decreased the release of a pro-inflammatory neuropeptide in the gut, and, most importantly, limited the amount of physical damage that occurred to the colon. These findings characterize the TRPM8 receptor as an anti-inflammatory receptor, and highlight its potential as a target for therapeutics directed at reducing inflammation in IBD and other chronic inflammatory diseases.
April 19, 2013
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Author’s note: I will be taking over writing duties from John Holyoake for the Friday Science Review. If you wish to know more about me, you can find my short bio on our contributors page – http://crossborderbiotech.ca/about/
Anxiety disorders are becoming increasingly prevalent in our society, and are highly detrimental to an individual’s well being, both physically and mentally. Children with autism spectrum disorder (ASD) are particularly susceptible to anxiety disorders, but their deficits in communication often make it difficult for them to express feelings of anxiousness. Being unable to express these feelings may lead to an exacerbated anxiety response in children with ASD, because they are often exceedingly aware of their surroundings and may become more socially withdrawn. A study published in PLoS One led by Dr. Azadeh Kushki at the Holland Bloorview Kids Rehabilitation Centre in Toronto sought to address this problem by determining a physiological marker that signals anxiety in children with ASD, which would allow for improvement in their care.
Children with ASD and typically developing children performed an anxiety-inducing task while activity of the autonomic nervous system, the part of our nervous system that unconsciously controls visceral functions, was evaluated using three different measures. The researchers found that two of these measures, heart rate and perspiration, were elevated even at rest in children with ASD, whereas the third measure, skin temperature, was comparable at rest between children with ASD and typically developing children. However, during the anxiety-inducing task the skin temperature of children with ASD increased, whereas the skin temperature of typically developing children decreased, the normal response during stress.
The results of this study offer three important findings. First, the observation that heart rate and perspiration are elevated at rest in children with ASD supports previous reports that these children have elevated generalized anxiety. Second, the difference in changes in skin temperature between children with ASD and typically developing children observed during the stressful task offer a potential non-invasive measure of anxiety in children with ASD. Finally, the generalized difference in visceral functions observed between children with ASD and typically developing children indicates that children with ASD experience inappropriate regulation of their autonomic nervous system, specifically over-activity in the division of the autonomic nervous system that controls stress responses. This final finding warrants further investigation in order to understand whether inappropriate regulation of the autonomic nervous system contributes to increased generalized anxiety in children with ASD, or if it simply accompanies general anxiety caused by other characteristics of the disorder.
February 15, 2013
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The Dick lab at the Campbell Family Cancer Research Institute has a prestigious history of unraveling the complexities of cancer and their recent publication in Science adds yet more detail.
Analysis of tumours has revealed that there is immense heterogeneity between patients and also between cancer cells within the same tumour. This heterogeneity is evident at the genetic level, but also in the morphology and behaviours of individual clonal cell lines.
In the research reported in Science, the Dick lab tracked the in vivo propagation of xenotransplanted patient-derived colorectal cancer cells at the resolution of individual clones and demonstrated that cancer cells with the same genetic lineage demonstrate significantly different functional states and notably included clonal cells that were dormant compared to their peers. These populations of dormant cells may be the source of disease recurrence after treatment and therefore determining the mechanism through which the cells enter and sustain their dormant state may open up a new therapeutic strategy to fully eradicate the disease and prevent recurrence.
January 11, 2013
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Ubiquitin-specific proteases (USPs) are implicated in many diseases and yet only a few weak inhibitors of their function have been available. A report in Science from a collaboration led by the Siddhu lab at the Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto describes the development of a molecular strategy to inhibit these important enzymes.
The conjugation of ubiquitin to proteins is one of the most common post-translational modifications of proteins. Labeling of these proteins by ubiquitin often functions to target the protein for destruction by the proteasome or to alter the proteins’ sub-cellular localization and activities. The labeling process is itself complex involving the sequential action of enzymes (E1, E2 and E3) and can result in the addition of a single ubiquitin or multiple ubiquitins either as a chain (polyubiquitination) or at multiple sites on the target protein (multiubiquitination). Removal of ubiquitin can rescue a protein from destruction or restore its activity and is carried out by deubiquitination enzymes, of which the ubiquitin-specific proteases (USPs) are the largest class. Members of this class are implicated in multiple disease processes, including cancer and neurodegeneration, making them an attractive area of study, but one that has been hampered by the lack of high affinity inhibitors.
USPs share a conserved ubiquitin binding site structure, albeit with low affinity for ubiquitin, therefore the research led by the Siddhu lab used the strategy of phage-display-based screening of ubiquitin mutants that had increased binding affinity for specific USPs. Using this approach several specific USP inhibiting mutants were created and the researchers were able to build upon this success by expanding the concept to inhibit other deubiquitinating enzyme classes and components of the ubiquitin labelling system – the E2 and E3 enzymes. In vivo activity of this system means the door is open to carry out detailed dissections of the ubiquitin system, along with using the system to validate drug targets, to create screening assays and ultimately to guide the generation of ubiquitin-system target specific inhibitors.
November 30, 2012
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Autism spectrum disorder (ASD) encompasses a range of developmental disorders that effect multiple basic functions including language and socialization. Specific causes of ASD have not been determined, though multiple risk factors have been identified, including genetic components and prenatal complications. Increased neuronal connectivity as a result of dysregulated and overactive synaptic protein expression is, however, proposed to be part of the molecular mechanism.
mTOR has previously been implicated in the observed overactive protein expression, but events downstream of mTOR have not been investigated. It is known that mTOR phosphorylates eukaryotic translation initiation factor 4E-binding proteins (4E-BPs), which are important for suppressing initiation of the translation of mRNA’s and therefore researchers at McGill University and the Université de Montréal investigated the effects of knocking out 4E-BP2 and of overexpressing eukaryotic translation initiation factor 4E (eIF4E). Their paper in Nature shows that both of these perturbations lead to overexpression of neuroligins, a postsynaptic protein class implicated in ASDs. Furthermore, the 4E-BP2 KO mice exhibited autism-like behaviours and had an altered excitatory-inhibitory synaptic balance. Returning neuroligin 1 levels to their normal range restored the synaptic balance, as did pharmacologic inhibition of eIF4E, opening up new avenues to explore for therapeutic interventions in ASDs.
November 9, 2012
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Histones are essential components of chromosomes, whose function is to both package DNA and to regulate the expression of genes. For the latter function, histones are subject to a range of post-translational modifications that can activate or silence transcription of the local DNA. The modification status of histones is determined by the activities of a range of enzymes that act to add or remove the modifications and the coordination of the different activities is essential to regulate the transcription or silencing of genes.
The recent PNAS paper from the Brand lab at the Ottawa Hospital Research Institute’s regenerative medicine program adds to our knowledge of this epigenetic process by determining the mechanism by which two proteins, H3K9 methyltransferase G9a/KMT1C and the H3K4 demethylase Jarid1a/KDM5A, work together to achieve developmentally regulated gene expression at the β-globin locus. Opening the window on these complex mechanisms will be key to developing new therapies for a wide range of conditions ah, cancer, autoimmune diseases and psychiatry.
October 5, 2012
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What happens to the genome of pathogenic bacteria when they are forced to adapt after being confronted by an antibiotic, or after colonizing a new host species? This was the question posed by the Kassen lab at Carleton University. In their study, published in PLoS Genetics, they exposed Pseudomonas aeruginosa, an opportunistic pathogen and the major pathogen in the lungs of cystic fibrosis sufferers, to antibiotics in CF-lung mimicking culture conditions and then used by whole-genome sequencing to follow the genomic basis of the bacteria’s adaptation.
As might be expected, they found changes in antibiotic resistance genes that correlated with the degree of antibiotic resistance of the particular experimental isolate. However, they also found other co-occurring mutations that played a role in antibiotic resistance, but that had different associated fitness costs, suggesting a wide range of compensatory mutations. Separately, the study was able to add to the body of evidence suggesting that cyclic-di-GMP signaling plays a role in the adaptation of the bacteria to CF-lung-like conditions – a signaling pathway that promotes the adoption of a biofilm phenotype rather than the motile planktonic phenotype.
Ultimately, the study shows that adaptation of an opportunistic pathogen to conditions reproducing its common host environment results in a mixture of expected and unexpected genetic adaptations. These adaptations represent new targets for therapies, but also illustrate the heterogeneity facing patients and clinicians as they try to address these infections in the clinic.
September 28, 2012
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Saccharomyces cerevisiae is a mainstay model organism for molecular biology, where dissection of its signaling pathways and interaction networks are used to build models that can be extrapolated to other higher organisms. Closer to home, S. cerevisiae shares many conserved features with other yeast species, such as Candida albicans, which can be opportunistic human pathogens. The virulence of C. albicans has been correlated to its ability to switch between budding yeast to filamentous forms, an ability that is shared by S. cerevisiae. Therefore, understanding the genes involved in the different morphologies of S. cerevisiae, which includes distinct forms of filamentation, can therefore lead to an increased understanding of the pathogenesis of opportunistic fungal infections – a growing problem amongst immunocompromised patients
In an international collaboration, led by the Boone lab at the University of Toronto, a global gene deletion approach was used to explore the genes required for the filamentous growth programs. In their paper published in Science, they show that unique genes appear to underlie each filamentation program, but that some key genes are important across filamentous growth. These core genes include MFG1 (YDL233w), whose gene product acts as a regulator of filamentous growth by binding to Flo8 and Mss11, transcription factors previously found to have morphogenetic roles. With the identification of MFG1 as a key regulator of filamentation, the potential for a novel therapeutic strategy to prevent the invasion of human tissues by filamentous fungal species like C. albicans arises.
September 21, 2012
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Left-sided congenital heart disease (LS-CHD) is one of the most commonly seen forms of cardiac malformations. Affected individuals suffer from a spectrum of cardiac issues that include bicuspid aortic valves, aortic valve stenosis, narrowing of the aorta and underdevelopment of the left side of the heart (hypoplastic left heart syndrome). Several lines of evidence indicate that LS-CHD is due to genetic factors, but the specific genetic causes are not currently known. An international collaboration, headed by the Andelfinger lab at the Université de Montréal, set out to explore the role of structural genomic variations by searching for copy number variants present in only affected individuals and not other family members.
Their study, reported in PLOS Genetics, revealed 25 new candidate genes for LS-CHD. The genes had diverse functions and included the SMC1A gene, involved in sister chromatid cohesion, MFAP4, believed to be involved in in cell adhesion or intercellular interactions, and CTHRC1, which is involved in vascular remodelling. Together it builds a picture that suggests broad alterations in angiogenesis may be the root cause of at least some of the incidences of LS-CHD. This work is part of the first steps in determining the detailed molecular pathophysiological mechanism of LS-CHD, an important part of understanding the diversity of patient outcomes and of developing therapies.
September 7, 2012
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This week saw the publication of two important papers on protein-protein interactions. The first paper, from a collaboration led by Andrew Emili at the University of Toronto, was published in the journal Cell and details a census of soluble protein complexes from human cells. In a second complementary paper, published in Nature and led by Jack Greenblatt also at the University of Toronto (and also involving their next door neighbours, the Emili lab), the first global, high-confidence physical interaction map of membrane proteins from Saccharomyces cerevisiae was developed.
Both of these studies produced a wealth of information and provide important resources to identify candidate disease genes, as well as to predict the function of individual proteins within their respective complexes. Indeed, the human cell census identified over 300 previously un-annotated protein complexes, that comprised over 1,000 proteins, some of which are have already been linked to human disease. Similarly the membrane protein study identified more than 1,700 membrane protein-protein interactions and 500 putative protein complexes that involved a membrane protein. On this later point, it is important to remember that membrane proteins represent the majority of drug targets, but have been notoriously difficult to study due to their amphipathic nature.
In addition to the identification of new protein complexes, both papers allowed the exploration of the evolutionary conservation of protein complexes across different species that emphasizes a generally high degree of conservation, but that could ultimately reveal the dangers and limitations of using simpler organisms as models of human diseases.
August 24, 2012
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Reduced blood flow to organs and peripheral tissue, as might be expected, leads to cell necrosis and death. The nature of the resulting ischemic disease depends upon the specific tissue affected and spans the range from acute conditions such as acute coronary syndrome or acute kidney injury, to chronic conditions such as peripheral artery disease and angina. Despite the development of both pharmacological and surgical treatment strategies, ischemic disease remains a leading cause of morbidity and mortality. As such, therapies that will reduce ischemia by promoting the regeneration of damaged vasculature are an active area of active research and cell-based therapies represent one avenue being pursued.
In a paper in Stem Cells, researchers at the University of Western Ontario show that by selecting for a rare population of aldehyde dehydrogenase expressing cells from human umbilical cord blood they could promote vascular regeneration in an animal model of ischemic disease. This rare population of cells (<0.5%) is enriched for early myeloid and stem cell-associated cells and displays a pro-angiogenic transcription profile, including the hallmark angiogenic signaling factors ANGPT1 and VEGFA. In vitro cell culture efficacy was demonstrated in experiments that showed that this cell population was able to promote human umbilical vein endothelial cell (HUVEC) survival and tube-like cord formation, both being indicators of pro-angiogenic activity. Interestingly, permanent engraftment of the cells was not required for the vascular regeneration, which increases the clinical development potential by potentially avoiding the need for long-term immune suppression of a patient receiving this allogeneic cell therapy approach.
August 3, 2012
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Antibiotic resistance is an increasingly pressing public health problem. While the prevalence of resistance is increasing, there is a simultaneous dearth of newly approved or in development drugs. For those that are in development the majority are natural products or derivatives thereof. Most of these represent improved variants of marketed compounds rather than a new mechanistic class, which increases the risk of the rapid appearance of resistance. The small pipeline and current lack of diversity therefore means the identification of novel antibiotics is an important task.
Cationic antimicrobial peptides (CAMPs) represent one such class and are produced by all living species. These peptides have varied structures, but share the feature of a segregation of cationic and hydrophobic resides and it is this gross physiochemical property that underlies the antibiotic effects. Multiple mechanisms can be involved in the bactericidal activity, including membrane disruption, binding of DNA and other cellular components. However, the ability to translate the promise of CAMPs into antibiotics is hampered by their pharmacokinetic properties and especially their susceptibility to degradation by serum proteases.
Various strategies are available to address the stability problems of potential peptide therapeutics and this week’s paper by the Schweizer lab at the University of Manitoba focuses on examining the potential of the incorporation of peptoid residues into CAMP’s to improve their stability. Encouragingly their peptoid analogues had similar activities to the parent peptides, again supporting the idea that CAMPs work through gross physical chemical properties rather than specific structural features. The next step for the researchers is to show that the retained activity is bolstered by improved serum stability.
July 20, 2012
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TDP-43 and its C. elegans ortholog, TDP-1 are DNA and RNA binding proteins that have multiple functions in repressing transcription, splicing pre-mRNA and regulating translation. TDP-43 is found to be a constituent of stress granules, cytosolic RNA/protein aggregates that form in response to cellular stress, be it from heat, oxidative or osmotic.
TDP-43 has also been found to be mutated and accumulated in multiple neurodegenerative disorders, including ALS (Lou Gehrig’s disease), frontotemporal dementia (a form of pre-senile dementia second in prevalence only to Alzheimer’s disease) and chronic traumatic encephalopathy (including dementia pugilistica).
Given TDP-1/TDP-43’s role in cellular stress response the Parker lab at the Université de Montréal asked whether TDP-1 participated in C. elegans’s cellular stress response and longevity pathways and whether it was through participation in the Insulin/IGF-signaling (IIS) pathway, a major axis of stress-response signaling and longevity in worms.
They found that the tdp-1 gene is responsive to stress and participates in the Insulin/IGF signaling pathway to regulate lifespan, as well as to respond to oxidative stress. While worms that lack tdp-1 were more sensitive to external stresses, over-expression of tdp-1 was itself toxic. Furthermore, tdp-1 expression was induced by the introduction of mutant TDP-43 due to increased oxidative stress and that this led to increase neuronal degradation and reduced lifespan. This leads to the model that tdp-1 was intended to protect from unfavourable conditions, but is now becoming aberrantly activated and is contributing to proteotoxicity and oxidative stress in the cell. If this malfunction is present in humans it may explain why TDP-43 is found to be associated with an increasing number of neurodegenerative conditions and would support the exploration of TDP-43 as a therapeutic target.
July 13, 2012
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Oncolytic viruses are a promising therapeutic approach that may finally be approaching the market; Amgen’s recently acquired OncoVEX GM-CSF is in phase III for melanoma with results expected in 2013 and Canada’s own Oncolytics has recently completed the first stage of its phase III trial for the treatment of platinum-refractory head and neck cancers.
For tumour types that are permissive to these viruses much of the promise arises from the specific replication in cancer cells and subsequent lysis. However, another activity that can help improve complete responses and prevent recurrence even when the virus has been cleared from the patient is the induction of antitumor immunity. This immunity is greatly stimulated by the viral replication and lysis process. Aiming to harness this effect, the recent paper in Molecular Therapy by researchers at the University of Ottawa describes an approach to generate a broad anti-tumour immunity against a multitude of tumour antigens through the use of an infected cell vaccine (ICV) platform, which is even applicable for tumours that are not permissive to oncolytic viruses.
The oncolytic VSV-Δ51 virus is able to induce a strong anti-tumour immunity in tumour cell lines, but only when viral replication occurs in the tumour cells. To overcome this, the researchers chose an ex vivo approach, whereby infection of isolated tumour cells with their oncolytic virus construct (the VSV-Δ51 virus, but expressing GM-CSF similar to the BioVEX approach) could be ensured in vitro by a high multiplicity of infection. A vaccine could then be prepared from this population of infected cells. With this approach mice were protected from subsequent tumour challenge and the induced innate and adaptive immune response was robust enough to control the growth of established tumours.
This approach offers a personalized vaccine comprising the full range of a patient’s tumour-specific antigens improving the hope for complete response and effective control of recurrence, albeit with a significant commercialization challenge arising from the inherently unscalable manufacturing, operational complexity and high production costs that is currently being faced by Dendreon’s Provenge.
July 6, 2012
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While there are a surprisingly low number of genes in the human genome, 95% of the multi-exonic genes are subject to alternative splicing and therefore the role of alternative splicing in permitting increased cellular and functional complexity should be more widely appreciated.
In different cell types and in response to different conditions, coordinated splicing regulation leads to the specific use of alternative exons and these regulated exon networks can be assumed to play important roles in specific processes and pathways – why else would it occur and be conserved. However, while this regulated behaviour is well known, there is a current lack of understanding of the functional effects imparted by the tissue-regulated alternative exons. A desire to fill in some of this gap motivated the research by a collaborative trio of labs at the University of Toronto. In their Molecular Cell paper, they show that proteins containing regulated exon networks tend to have more interactions in protein-protein interaction networks and that these regulated exons, together with their flanking constitutive exons, are enriched in sequences predicted to be highly disordered.
In a specific example of an alternative exon network regulated by nSR100/SRRM4 (neural-specific Ser/Arg-repeat related protein of 100 kDa), which is responsible for the inclusion of ~11% of brain specific exons, they show that the inclusion of the regulated exons could result in more or fewer protein-protein interactions, depending on the specific gene. Therefore, introduction of a regulated exon and the corresponding additional protein segment does not merely have an additive effect on the range of interactions in which an alternatively spliced protein can engage. Continuing on to a deeper level of detail, the authors show that the inclusion of one of these regulated exons in Bin1 promotes the interaction with the GTPase Dnm2 and that this interaction was needed for efficient endocytosis in neural cells, linking the splicing regulation all the way to cellular function.
Changes in exon splicing are known to be both causes and consequences of multiple human diseases, including tauopathies, spinal muscular atrophy and familial dysautonomia; adding to the functional understanding of alternative splicing will surely allow the identification of more, and the increased chance for the development of a treatment.
June 22, 2012
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The Dick lab at the Campbell Family Institute of the Ontario Cancer Institute has been instrumental in the field of cancer stem cells. The central tenet of the cancer stem cell hypothesis is that there is a small population of cells within a tumour that are able to self-renew and also differentiate into the full range of cell types found within that tumour. In addition to their role at the head of the tumour cell hierarchy, these cancer stem cells (also known as cancer initiating cells, or tumour initiating cells) have increased resistance to chemotherapeutics, leading to an unfortunate ability to cause both relapse and metastasis.
In their Cancer Cell paper, the Dick lab and collaborators investigated the genes that are involved in controlling this combination of abilities. They determined that the proteins ID1 and ID3 were responsible for controlling the self-renewal of colon cancer stem cells. ID1 and ID3 are members of the DNA-binding protein inhibitor family of proteins that heterodimerize with basic helix-loop-helix transcription factors to prevent their ability to bind DNA. ID1 and ID3 appeared to be exerting their effect by increasing levels of p21, a cell-cycle inhibitor and regulator of self-renewal. p21 itself has previously been linked to protecting cancer cells from stress and DNA damage and it was also found that knocking down ID1 and ID3 led to increased sensitivity to oxaliplatin, thereby linking the phenomenon of increased resistance to chemotherapy and self-renewal.
June 1, 2012
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The cancer stem cell (CSC) hypothesis – the concept that a small population of stem cell-like transformed cells is important for cancer initiation, progression and relapse – has gained quite a bit of attention in both academia and industry. It is proposed that these populations of cells have the unfortunate combination of being able to reinitiate tumour growth following treatment, as well as being more resistant to treatment, which explains the development of therapy resistance, minimal residual disease and tumour recurrence.
An attractive therapeutic rationale is therefore to develop drugs that either selectively target the cancer stem cells. However, any such treatment would need to have a therapeutic window between killing the cancer stem cells and killing an individual’s healthy stem cells, a population of cells that play important roles in tissue maintenance and repair. One approach is to discourage the proliferative self-renewal role that CSC’s favour and encourage their differentiation, thereby reducing their tumour growth potential. However, despite exactly this mechanism being used by Vesanoid, a treatment for acute promyelocytic leukemias developed over 20 years ago, no similar treatments for other cancers exist. However, the research by the Bhatia lab and collaborators at McMaster University is aimed at reinvigorating this approach.
They developed a discovery platform that can be used to screen molecules for their ability to induce the differentiation of a model of human CSC’s (a variant human pluripotent stem cell line) and but not normal pluripotent human stem cells. Applying their platform in a proof-of-concept screen, initially with well established, annotated compounds from the NIH Clinical and Canadian Compound collections they identified several candidates that induced morphological changes in the CSC model cells, but had no effect on normal stem cells. Unexpectedly a compelling hit arose from the antipsychotic drug thioridazine, with the apparent activity arising from the molecules action as a dopamine receptor antagonist.
With a paper that presents both a powerful screening tool, a potential repurposing of an approved molecule and a new potential target for the selective treatment of CSC’s, there is certainly no shortage of avenues to pursue and hopefully it will not be another 20 years before the we see the addition of another cancer therapy using the differentiation approach.
- Divergent Genomic and Epigenomic Landscapes of Lung Cancer Subtypes Underscore the Selection of Different Oncogenic Pathways during Tumor Development. PLoS One. British Columbia Cancer Research Centre
- Inhibition of Serine Palmitoyl Transferase I Reduces Cardiac Ceramide Levels and Increases Glycolysis Rates following Diet-Induced Insulin Resistance. PLoS One. University of Alberta
- Assessment and implication of prognostic imbalance in randomized controlled trials with a binary outcome – a simulation study. PLoS One. McMaster University
May 25, 2012
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Most people take for granted that given a little resolve at the gym they can induce their muscles to grow, however, most people have probably not considered the complexity of the underlying cellular and biochemical processes. In response to exercise or injury, normally quiescent muscle satellite cells are activated and divide to produce progeny myogenic precursor cells that will themselves undergo multiple rounds of division before differentiating and fusing to the multinucleated muscle myofiber, thereby increasing the size and strength of the muscle. Key to the regulation of the satellite cell function is expression of the paired box transcription factors Pax3 and Pax7. These transcription factors are highly related (>85% sequence identity) and play overlapping, but mostly nonredundant roles in the specification and progression of the adult satellite cell lineage. Lineage tracing has suggested that Pax3 is required in cells that contribute to embryonic myoblasts and to the endothelial lineage, but Pax7 cells contribute to fetal myoblasts.
Given their sequence similarity, researchers at the University of Ottawa investigated how the functional differences were achieved. By profiling the global gene expression of satellite cell-derived myoblasts, alongside determining the genome-wind binding sites of Pax3 and Pax7, they showed that Pax3 and Pax7 have intrinsic differences in DNA binding and it is the differential binding that drives the differential downstream gene activation. Specifically, they showed that Pax3 and Pax7 are both able to activate gene expression by binding to combined prd/hbox motifs, but Pax7 can also activate gene expression by binding to the hbox motif alone. Due to this difference in binding ability, over 400 genes are regulated by Pax7, that are not subject to regulation by Pax3 and these genes have diverse functions from cell adhesion to muscle cell differentiation. This work adds to the transcriptional network underpinning muscle cell differentiation and also cautions us by showing how large functional differences can occur in transcription factors with only small differences in sequence.
- Hyperphosphorylation and cleavage at d421 enhance tau secretion. PLoS One. Université de Montréal
- Structural basis for substrate specificity and catalysis of human histone acetyltransferase 1. PNAS. Structural Genomics Consortium, Toronto
- Muramyl Dipeptide Induces NOD2-Dependent Ly6C(high) Monocyte Recruitment to the Lungs and Protects Against Influenza Virus Infection. PLoS One. Laval University
May 11, 2012
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The tumour suppressor p15ink4b is a cyclin-dependent kinase (cdk) inhibitor, which functions to cause cell cycle arrest and whose functional presence in tumour cells is often lost through mutation or deletion. The expression of p15ink4b can be rapidly induced by transforming growth factor beta (TGF-β) and regulation of p15ink4b levels occurs primarily at the level of transcription. DNA methylation is one of the mechanisms known to repress transcription of p15ink4b, however, the mechanism by which DNA methylation is reversed for the regulation of transcription is relatively poorly understood. In this week’s Molecular Cell paper, the Torchia lab at the University of Western Ontario sought to determine the mechanism of DNA methylation and demethylation of the p15ink4b in response to TGF-β.
Using chromatin immunoprecipitation-sequencing (ChIP-seq), they showed that the p15ink4b gene is a target for the ZNF217/CoREST complex and that along with the action of specific DNA (cytosine-5)-methyltransferase enzymes e.g. DNMT3A, the p15ink4b gene is hypermethylated and repressed. Overcoming the repression in response to TGF-β induced signaling was shown to require removal of the DNMT3A/ZNF217/CoREST complex and replacement by SMAD2/3, the CBP acetyltransferase, and TDG or MBD4. Base excision repair then occurred, demethylating the DNA and thereby removing the transcriptional repression. They further showed that ZNF217 overexpression, a feature of some cancers, was shown to inhibit recruitment of the demethylation complex.
While focused on the specific regulation of p15ink4b, these results add more generally to our knowledge of methylation-based epigenetic regulation and the important association of abnormal DNA methylation patterns with malignant transformation.
- Monomeric site-specific nucleases for genome editing. PNAS. University of Western Ontario
- TRADD contributes to tumour suppression by regulating ULF-dependent p19(Arf) ubiquitylation. Nature Cell Biology. University of Toronto
The innate immune system is the first line of defense against infection of the host organism by pathogens and in this system, type I interferons are a critical component in limiting viral replication. Indeed, type I interferons are already marketed or in development for their anti-viral and immune modulating activities.
Levels of type I interferons are subject to control and rapid induction in response to the recognition of a pathogen by the host organism’s pattern-recognition receptors (e.g. Toll-like receptors – topically a struggling area of development with Idera’s recent phase II failure of a TLR9 inhibitor for advanced head and neck cancer). Production of the type I interferons is rapidly stimulated through a cascade that includes regulation of both transcription and translation, with the transcriptional activator NF-kB being an important site of regulation. A paper in Nature Immunology from a international consortium of researchers led by the Sonenberg lab at McGill University presents data showing that phosphorylation of the translation factor eIF4E is a key mechanism in regulating NF-kB’s ability to activate type I interferon production. Using mice in which the Ser209 phosphorylation site in eIF4E is removed, they showed indirect enhancement of NF-kB activity through decreased translation of NF-kB’s inhibitor IkBa. Furthermore, they showed that in these mice the replication of three different RNA viruses was impaired and that the mice were more resistant vesicular stomatitis virus (VSV) infection associated neurological damage.
Currently Mnk1 and Mnk2 are the only known kinases to phosphorylate eIF4E in mice, with Mnk2 being constitutively active and Mnk1 being activated in response to mitogens, growth factors and hormones, which places Mnk1 in the cascade initiating response to pathogen detection. Earlier this year researchers at the Child and Family Research Institute at the University of British Columbia identified Mnk1 as mediating Trastuzumab (Herceptin) resistance in HER2-positive breast cancers, which excitingly identifies Mnk1 as an unexploited potential target for multiple therapeutic categories, including cancer, viral infections and immune modulation.
- A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities. PNAS. University of British Columbia
- Innate immune response to rift valley Fever virus in goats. PLoS Negl Trop Dis. National Center for Foreign Animal Disease, Canadian Food Inspection Agency
- Trappin-2/Elafin Modulate Innate Immune Responses of Human Endometrial Epithelial Cells to PolyI∶C. PLoS One. McMaster University
- Inflammation-Driven Reprogramming of CD4(+)Foxp3(+) Regulatory T Cells into Pathogenic Th1/Th17 T Effectors Is Abrogated by mTOR Inhibition in vivo. PLoS One. McGill University
- Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN. PLoS One. University of Western Ontario
- Early steps in oxidation-induced SOD1 misfolding: Implications for non-amyloid protein aggregation in familial ALS. J. Mol. Biol. University of Toronto
April 27, 2012
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All vertebrates share a general brain structure, featuring a forebrain, midbrain, and hindbrain, something that is most evident during embryogenesis. However, it is the size and structure of the forebrain and most notably the cerebral cortex that differentiates mammals from the other vertebrates.
As might be expected, the process by which the mammalian forebrain develops is a hugely complicated and fortunately tightly regulated process that involves the expansion of neural progenitor cells, followed by coordinated asymmetric divisions and differentiation to produce the specialized neuronal subtypes that make up the six layers of the cortex.
Regulation of this process is achieved through a combination of internal cellular programs and modulation by external signaling factors. Neurogenic transcription factors were obvious actors in the intrinsic cellular programs, however, observations that mutations in genes encoding chromatin remodeling proteins caused neurodevelopmental disorders has implicated a role for epigenetics in brain development. It is to our understanding of this epigenetic mechanism to which researchers at the Ottawa Hospital Research Institute and the University of Ottawa have contributed with their recent paper in Developmental Cell.
They sought to determine the in vivo roles of the mammalian ISWI chromatin remodeling protein Snf2l in murine forebrain development by using a conditional targeting approach to remove Snf2I’s ATP-binding motif, thereby impairing its chromatin remodeling activity. Mice containing the mutated Snf2I showed enhanced neural progenitor cell expansion and forebrain hypercellularity that arose from an increased progenitor cell cycle rate and enhanced self-renewal. The hypercellularity was caused by perturbation of the progenitor cell cycle kinetics and delays in the initiation of differentiation and neurogenesis. Expression profiling of the mutant mice, identified increased expression of Foxg1/Brain factor-1, a forkhead homeodomain transcription factor, and the researchers were able to show that Snf2I’s normal role is to reduce Foxg1/Brain factor-1 expression, thereby resulting in the increase in activity of p21 and terminal differentiation of the progenitor cells.
- Structure of an intermediate state in protein folding and aggregation. Science. University of Toronto
- Dysregulation of cell polarity proteins synergize with oncogenes or the microenvironment to induce invasive behavior in epithelial cells. PLoS One. University of Toronto
April 20, 2012
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This week’s Friday science review covers a Nature paper from a large international collaboration – 19 affiliated institutes no less – with the lead author from the University of British Columbia/British Columbia Cancer Research Centre. They present results from the study of somatic mutations in triple negative breast cancer (TNBC) patients.
TNBCs are a subset of breast cancers (~16%) that do not express the estrogen receptor, progesterone receptor or HER2; cannot be treated with endocrine therapy or the HER2-targeting Herceptin (trastuzumab); and are associated with relatively poor outcomes. They show that at the time of diagnosis for 104 TNBC cases the tumours exhibited a wide and continuous range of genomic evolution spanning from a few somatic alterations to hundreds of somatic genomic aberrations. Looking deeper in to the clonal variation within an individual tumour they found significant differences, which meant that at diagnosis the TNBCs already displayed a widely varying clonal evolution. As expected, known drivers such as p53, PIK3CA and PTEN were amongst the highest clonal frequencies, indicating that they were early events in the clonal evolution, however, this was not always the case and in some cancers another unidentified driver of early clonal expansion must have been present.
Ultimately this work adds to the growing sense that to really understand the mechanics driving tumour growth and to predict therapeutic responses will require looking into the individual tumour clonal genotypes.
- Gastrointestinal-sparing effects of novel NSAIDs in rats with compromised mucosal defence. PLoS One. McMaster University
- Aberrant Localization of FUS and TDP43 Is Associated with Misfolding of SOD1 in Amyotrophic Lateral Sclerosis. PLoS One. University of British Columbia
- TOR Is Required for the Retrograde Regulation of Synaptic Homeostasis at the Drosophila Neuromuscular Junction. Neuron. McGill University
- Engagement of β-arrestin by transactivated insulin-like growth factor receptor is needed for V2 vasopressin receptor-stimulated ERK1/2 activation. PNAS. Université de Montréal
- Road crash fatalities on US income tax days. JAMA. University of Toronto