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Category Archives: John Holyoake

Friday Science Review: February 15, 2013

John HolyoakeThe 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.

Friday Science Review: January 8, 2013

John HolyoakeUbiquitin-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.

Friday Science Review: November 30, 2012

John HolyoakeAutism 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.

Friday Science Review: November 9, 2012

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.

Friday Science Review: October 5, 2012

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.

Friday Science Review: September 28, 2012

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.

Friday Science Review: September 21, 2012

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.

Friday Science Review: September 7, 2012

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.

Friday Science Review: August 24, 2012

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.

Friday Science Review: August 3, 2012

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.

Friday Science Review: July 20, 2012

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.

Friday Science Review: July 13, 2012

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.

Friday Science Review: July 6, 2012

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.

Friday Science Review: June 22, 2012

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.

Bloom Burton & Co. Open the TSX

Leading up to their inaugural healthcare conference later this week, Bloom Burton & Co opened trading on the TSX this morning. In a feat of multimedia wizardry, the moment was captured on film:

Friday Science Review: June 1, 2012

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.

Other publications:

  • 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

Friday Science Review: May 25, 2012

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.

Other publications:

  • 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

Friday Science Review: May 11, 2012

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.

Other publications:

  • 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

Friday Science Review: May 4, 2012

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.

Other publications

  • 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

Friday Science Review: March 2, 2012

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. However, current treatments e.g. dopamine precursors, monoamine oxide inhibitors and COMT inhibitors, do not aim to address the underlying loss of dopamine containing neurons, but rather, most focus on modulating the extant dopamine metabolism.

In studying PD, familial PD cases have been useful in adding to the understanding of pathophysiology. Importantly, they revealed the association of mutations in mitochondrial proteins with PD, thereby implicating mitochondrial dysfunction in the causes of the disease. One of the identified mitochondrial proteins, PTEN-induced putative kinase 1 (PINK1) is usually trafficked to the interior of mitochondria, where it is rapidly degraded. However, in dysfunctional mitochondria, the reduction in proton electrochemical gradient is associated with the failure of PINK1 to traffic to the mitochondrial interior. Instead, PINK1 accumulates on the surface of the cytoplasmic face of the outer mitochondrial membrane, where it recruits another familial PD-implicated protein Parkin. It is the recruitment of Parkin to the depolarized mitochondrion that sets in motion mitophagy and clearance of the dysfunctional mitochondrion. As such, the PINK1-Parkin pathway represents an important quality control mechanism, helping cells to remove dysfunctional mitochondria and the deleterious effects they cause due to the release of reactive oxygen species or pro-apoptotic factors.

A key question in this mechanism becomes, what is regulating PINK1? The article by the Fon lab at McGill university helps answer part of this question. By selectively screening mitochondrial proteases using RNA-mediated interference, they were able to identify mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP as being involved in the degradation of PINK1. Of these, MPP appears to be the major factor in PINK1 turnover, with even slight reductions in MPP activity resulting in PINK1 accumulation at the mitochondrial surface. The authors suggest that potentiation of the PINK-1-Parkin pathway, possibly by low level inhibition of MPP, could be an avenue to explore in treating PD by increasing the stringency for mitochondrial function and thereby reducing oxidative stress in dopaminergic neurones. While a single eukaryotic cell has over 1000 mitochondria, one has to assume that altering the fundamental balance of mitochondrial turnover is going to be fraught with perils.

However, as highlighted by the rather sparse KEGG PD pathway map, all advances that elucidate the proteins and events in PD are vital in the search for a unified, global understanding of PD, be it familial or idiopathic, and the hope for a therapy that can address the underlying molecular mechanism.

Other Publications

  • Repression of a Potassium Channel by Nuclear Hormone Receptor and TGF-β Signaling Modulates Insulin Signaling in Caenorhabditis elegans. PLoS Genetics. University of British Columbia
  • Charged and hydrophobic surfaces on the a chain of shiga-like toxin 1 recognize the C-terminal domain of ribosomal stalk proteins. PLoS One. University of Toronto
  • Lack of functional selectin ligand interactions compromises long term tumor protection by CD8 T cells. PLoS One. University of Ottawa

Friday Science Review: February 24, 2012

In my first contribution to the Cross-Border Biotech Blog’s Friday Science Review we have a promising advance in the treatment of Huntington’s disease by an international collaboration led by researchers from the Department of Pharmacology at the University of Alberta and another look at the surprisingly small number of critical genes in organisms by the Fraser lab at the University of Toronto.

A promising new avenue for Huntington’s Disease

The autosomal dominant genetic disorder Huntington’s disease (HD) is caused by the expansion of the CAG codon in the Huntingtin gene and the resulting inclusion of an abnormally long poly-glutamine stretch in the Huntingtin (Htt) protein. The expanded poly-glutamine stretch results in misfolding of the Htt protein and the formation of aggregates that are deposited as inclusion bodies within cells. As Htt protein is most highly expressed in neuronal cells, the aggregates lead to impaired neuronal transmission and ultimately to neuronal death, resulting in the loss of muscle coordination, cognitive impairment and psychiatric problems that are characteristic of HD.

Current treatments for HD are palliative in nature only, however, the paper by Di Pardo et al, published in PNAS, aimed to address the molecular mechanisms of HD by intraventricular infusion of the ganglioside GM1 – for those of us that like to picture molecules, GM1 is a glycosphingolipid with a headgroup attached sialic acid. GM1 levels have previously been shown to be reduced in HD animal models and post mortem HD patient brain samples. In this study, they show that infusion of GM1 reduces Htt toxicity and restores normal motor function in symptomatic HD mice models. GM1 appeared to exert its effect, by inducing an increase in DARPP-32 levels and phosphorylation, as well as by inducing the phosphorylation of the Htt protein at specific serine residues that reduce Htt toxicity.

While there is the chance that the rather invasive GM1 approach itself might represent a therapy, at the least, these results suggest new therapeutic pathways to research and target for the treatment of HD.

Eukaryotic genes: Necessary, but not required?

Previous studies in a range of model organisms have presented us with something of a paradox: eukaryotic genomes are highly conserved indicating that most genes are functionally important, yet only a minority of genes have detectable loss-of-function phenotypes. Models explaining this paradox range from suggestions that genetic networks have evolved to be robust and resistant to individual mutations, to suggestions that the targeted genes are essential, but just don’t happen to be required in the particular conditions used in the experiments.

The Fraser lab at the Donnelly Centre of the University of Toronto has helped resolve this paradox by showing in last weeks issue of Cell that, in fact, the majority of genes in C. elegans, if individually suppressed by RNA mediated interference, result in lower multi-generational fitness compared to wild-type. This result challenges the model that genetic networks are robust and instead suggests that the loss of most genes results in phenotypes that were too subtle to have been detected by previous assays. The complex interactions and subtle phenotypes, that manifested as decreased fitness, emphasizes that the development of a systems level understanding of gene function will be increasingly important to understand the molecular basis of diseases.

Other Publications

  • SKI-1 and Furin Generate Multiple RGMa Fragments that Regulate Axonal Growth. Developmental CellToronto Western Research Institute ♦ University of Toronto
  • Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID-Mediated Mutagenesis. PLoS Genetics. University of Toronto
  • Genetic variation in cell death genes and risk of non-hodgkin lymphoma. PLoS One. Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency
  • Caffeic Acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes. PLoS One. Université de Moncton

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