The Cross-Border Biotech Blog

Biotechnology, Health and Business in Canada, the United States and Worldwide

Friday Science Review: June 24, 2011

New Drug Targets in the Ubiquitin-Proteasome System?

Mount Sinai Hospital ♦ University of Montreal ♦ Structural Genomics Consortium ♦ Celgene Signal Research Division ♦ Wellcome Trust Centre for Cell Biology ♦ University of Toronto

Published in Cell, June 24, 2011

Proteins within the ubiquitin-proteasome system are responsible for modulating the stability and cellular localizations of a plethora of proteins in the human body. Usually it is the E2 enzymes that pass ubiquitin along to a variety of substrates to alter their stability. Researchers have discovered a small molecule, being coined C60651, that latches into a cryptic sight on the E2 enzyme hCdc34, changing it structural conformation and ability to transfer molecules of ubiquitin to receptor lysine residues on target substrates. Further investigation with C60651 showed that it could stunt the growth of human cancer cell lines. Presumably, the small molecule broadly affects protein stability and interferes with essential cellular processes. Thus, it could be worth investigating the deactivation of E2 enzymes as a therapeutic paradigm for cancer.

JAK2 at the Heart of Chuvash Polycythemia

University of Toronto ♦ University of North Carolina ♦ University of Pennsylvania ♦ UC San Diego ♦ Hospital for Sick Children

Published in Nature Medicine, June 19, 2011

Chuvash Polycythemia is a rare congenital disorder characterized by an increase in the total number of red blood cells. It results primarily from genetics defects in erythroid progenitors that make them either hypersensitive or insensitive to stimulation by erythropoietin. This recent study illustrates that regulation of JAK2 could be at the centre of the disease. Typically, homozygous mutations in the R200W and/or H191D genes located in the von Hippel-Lindau (VHL) gene cause the disease. Researchers show that under normal circumstances the VHL gene product binds to suppressor of cytokine signaling 1 (SOCS1) to form a dimeric ligase that marks JAK2 for ubiquitin-mediated degradation. However, after analyzing the VHL gene product in Chuvash Polycythemia, it was found that the mutant version fails to form a complex with SOCS1 and hence fails to elicit the destruction of JAK2. To substantiate their findings, investigators treated knock-in mice homozygous for the R200W mutation with a JAK2 inhibitor and showed that the disease phenotype could be reversed.

Friday Science Review: June 17, 2011

New Players in Interleukin-17 Response to Bacterial Pathogens

University of Toronto ♦ St. Michael’s Hospital ♦ Published in Nature Medicine, June 12, 2011

Interleukin-17 (IL-17) is a well established chemical messenger that modulates antimicrobial immune response in the stomach and intestine in the presence of various bacterial pathogens, including Pseudomonas, HelicobacterCitrobacter, and Salmonella; this has been known for some time, but the mechanism stimulating IL-17 release was until now a mystery. Researchers recently discovered that in the presence of such pathogens, CD4+ T helper cells interact with TGF-β and IL-6 to differentiate into T helper type 17 (Th17) cells that are characterized by the secretion of IL-17 and IL-22. Furthermore, they found that Th17 cells are regulated by the Nod-like receptors, Nod-1 and Nod-2. Researchers termed the cells “innate” T helper type 17 cells due to their early induction and regulation by Nod receptors, unlike adaptive-phase Th17 cells that arise at late stages of exposure. As a result of their involvement in regulating inflammation and antimicrobial response, it is believed that the Nod receptors may play a role in inflammatory bowel disease.

Treating Atherosclerosis: Shipping the Cholesterol Out of Foam Cells

University of Ottawa Heart Institute ♦ Published in Cell Metabolism, June 8, 2011

Macrophage foam cells contain large quantities of cholesterol inside an organelle known as a lipid droplet. Macrophages rich in cholesterol are problematic, as they tend to build up in atherosclerotic lesions causing plaque build-up in the arteries. An interesting paradigm for the treatment of atherosclerosis has been reverse cholesterol transport; the process wherein cholesterol hidden away within lipid droplets is hydrolyzed and removed from the peripheral tissues to the liver, where it can be excreted via bile. Over the years researchers have been investigating how cholesterol hydrolysis actually occurs to assess whether it can be leveraged to treat atherosclerosis. The dominant theory has been that all hydrolysis is driven by neutral cholesterol hydrolases, but new findings show that another mechanism also contributes to the process. Researchers found that lipid droplets in the cytoplasm of foam cells are transported to another organelle, known as a lysosome, where they are absorbed and broken down by lysosomal acid lipase. Lipid droplets are delivered to lysosomes while additional cholesterol is being loaded into the cytoplasm of macrophages from the blood, suggesting that lysosomal degradation serves as a reverse cholesterol transport mechanism.

Preview — BIO and Scientific American’s Regional Bio-Innovation Scorecard

The BIO 2011 conference is just around the corner, and Washington DC prepares for some 15,000 delegates from 65 different countries to descend upon its limits, which means it’s almost time for this year’s Worldview Regional Bio-Innovation Scorecard. This morning, the Biotechnology Industry Organization (BIO) hosted a press conference in DC to provide some highlights on the upcoming conference and to leak a few details of what this year’s scorecard would capture.

Over the last three years, Scientific American has partnered with BIO to produce a global ranking of countries in several areas related to innovation in the biotechnology sector (find a link to the 2009 results here). Jeremy Abbate, Director of Global Media Publishing at Scientific American, explained that as certain countries around the world are catching up to the US in terms of their production of innovative products, the biotechnology sector is becoming exceedingly global, and that a publication like Scientific American Worldview: A Global Perspective is needed to broadly document new trends and developments.

The essence of Worldview is to promote dialogue on what elements drive innovation, and to answer some pertinent questions: what characteristics or attributes make a country innovative? what makes a person innovative? Another goal is to identify individuals that are forces within the sector — those influential characters that are making the difference — the so called “heroes of science and finance”. Abbate iterated that although Worldview does not have all of the answers yet, it has collected massive quantities of data on the biotech sector in pursuit of them, and the scorecard is its “pièce de résistance”.

So what can you expect to see in 2011’s Worldview Regional Bio-Innovation Scorecard?

Emerging Markets

Last year’s separated the hype from the hope with a focus on China. This year China will be under the spotlight again, but this time along with the first three of the BRICs countries, including Brazil, Russia, and India. The scorecard will look closely at what these countries have to offer in terms of their capacity to innovate, their scientific output, and what capital they have available for investment. A special emphasis will be placed on the bio-innovation workforce they have to deploy across the biotechnology, medical device, and pharmaceutical industries.

The 2011 scorecard will point to the world’s fastest growing drug markets, take a deep dive on Africa and Australia, and present some analysis on innovative products, what countries pay to produce them, and how price has been affecting output. Russia’s Pharma 2020 initiative also comes into play. Will Russia be the manufacturing powerhouse that some hope it will be? During the briefing, Nikolay Savchuck, passed industry representative to the Russian government’s Pharma 2020 initiative, joked with the press that, unlike China, Russia has been all hope and no hype; perhaps it is Russia’s turn to deliver.

Biotech Movers

Making its debut to the scorecard will be the “Worldview Biotech Movers”. This section will highlight three game changers in the biotech sector and what they are doing to lubricate the cogs of innovation. This year expect to become a little more familiar with India’s Kiran Shaw of Biocon, China’s Ge Li of WuXi AppTech, and one other influential individual from Brazil. Although not always “friendly” to innovation on a global scale, these people march to their own drum and thrive on a regional basis.

The movers section of the scorecard touches on Abbate’s question — what makes a person innovative? Unlike definable elements that contribute to a country’s capacity to innovate, like intellectual property, a sound regulatory environment, science-minded policy, R&D investment, and so forth, the qualities of a great human innovator are much more nebulous. Ultimately, a country’s capacity to innovate may rely to a large degree on people. The work behind Worldview may in time bring some tangibility to this subject.

Snapshot from Canada

Cate McCready, VP of External Relations at BIOTECanada, spoke to the press and provided a snapshot of the Canadian biotechnology sector. In 2008, the Canadian government began an industrial analysis of biotechnology in the country which indicates the sector is contributing in the realm of $86 billion annually to Canada’s GDP. Although this figure is primarily driven by healthcare, McCready notes that there has recently been a divergence to other biotechnology industries, namely agricultural biotechnology, which is growing rapidly. Canada currently has 600 biotechnology companies operating within its borders, from “coast-to-coast-to-coast”, as McCready puts it. Be sure to check out Canada Cafe at the conference later this month.

A Few Highlights

So which countries will win accolades? The scorecard preview didn’t give up many hard stats, and leaves much to the imagination, so you’ll have to wait until it’s unveiled at BIO on June 29th.

Officially in, however, Denmark will take no. 1 for biotech patenting, while New Zealand proves to have the greatest number of PhD candidates per capita.

Friday Science Review: June 10, 2011

Silence is Golden

University of British Columbia ♦ BC Cancer Agency ♦ Institute for Virus Research ♦ Kyoto University

Published in Cell Stem Cell, June 3, 2011

Researchers analyzing the genome wide repression of genes and other repetitive elements, like endogenous retroviruses (ERVs), have come upon some findings that illustrate how epigenetic silencing occurs in mouse embryonic stem cells (mESCs). Epigenetic silencing of genes typically serves to repress the expression of very specific portions of DNA, and it has also been known to repress snippits of viral DNA found in the long winding portions of DNA between genes. To analyze what would happen in the absence of epigenetic silencing, researchers genetically modified mESCs to be null for DNA methyltransferase and the H3K9 methyltransferase Setdb1 — two enzymes that are very active in gene silencing via the deposition of methyl groups to DNA (preventing transcription of genes). Interestingly, as opposed to observing an overlap in the genetic elements that were activated in the absence of these enzymes, researchers found that two very distinct sets of genes were activated. About 15% of the genes activated in the absence of Setdb1 were activated by the promoter of an endogenous retrovirus. In as much as half of these cases, viral transcripts were fused to regular gene transcripts producing chimeric transcripts. These findings provide a role for the methyltransferase Setdb1 in silencing retro-elements and repressing aberrant transcriptional events that could lead to potentially harmful proteins downstream.

Inflammation, Innate Immunity, and the Intestine

McGill University ♦ Published in Nature, June 2, 2011

A key mechanism by which the intestine protects itself from the threat of foreign-invaders, the likes of pathogens and other microbes, is through the nucleotide-binding and oligomerization domain (NOD) proteins located in the interior of intestinal cells. These pattern recognition receptors have been highly conserved throughout the evolution of the innate immune system due to their importance in sensing infection and other dangerous signals. NOD proteins have the ability to sense proteins on the extremity of microorganisms and in response elicit a signaling cascade leading to the release of chemical messengers known as cytokines that drive an immune response and inflammation. Until recently , the exact nature of the signaling mechanisms controlling this process were unknown but as a result of some work with RNAi on the genome scale we have a much better understanding. Researchers at McGill conducted a small interfering RNA screen of 7170 human “druggable” genes to identify candidate genes that modulate the NOD inflammatory response. In doing so they identified the protein BID as an activator of NOD signaling. Macrophages from mice lacking the BID gene are highly defective in cytokine production while the mice themselves are unresponsive to local or systemic exposure to molecules that activate NOD signaling. Interestingly BID has a key role in programmed cell death, strengthening the already existing theory that programmed cell death and immunity are linked on some level.

Friday Science Review: June 3, 2011

Horizontal Gene Transfer: Bacterial Adaptation

McGill University ♦ Fudan University, China ♦ Published in PLoS ONE, May 20, 2011

The bacterium S. aureus can cause a host of problems in humans, companion animals, and cattle. Its ability to adapt and circumvent the effects of antibiotics allow it to persist, while virulence factors, acquired via horizontal gene transfer (HGT) with surrounding microflora, give S. aureus the capacity to cause disease relatively easily. The mobile genetic elements that allow for HGT to occur can come in different fashions, including viruses, plasmids, and other elements that may be self-transmissible or piggy-back with mobile viruses and plasmids. By comparing the genetic content of different strains of S. aureus researchers were able to evaluate sequence diversities from different sources to measure the extent that HGT contributes to genetic diversity. Comparative genomic analyses of strains derived from cows, sheep, chickens, and humans revealed that they are all highly associated with one another. Although the Serine-Aspartate Repeat (Sdr) family proteins in S. aureus are of unknown function, they have previously been correlated with human disease. Researchers were able to type S. aureus with regional differences in sdr gene distribution and make associations between specific distributions and clinical samples. At least one new insertion sequence observed was responsible for the HGT that allowed the sdrC gene to migrate between strains.

Eggs from Skin

University of Guelph ♦ University of Western Ontario ♦ Queen’s University

Published in PLoS ONE, May 19, 2011

A group that previously isolated stem cells from fetal pig skin has isolated multipotent stem cells from mouse skin and shown that they can differentiate into oocyte-like cells in vitro and in vivo. Researchers used a flourescent marker linked to the gene OCT-4 under the control of a germ cell-specific DNA enhancer element to see if germ cells would arise from mouse stem cells upon differentiation in culture. After cells were allowed to differentiate, a small fraction were GFP+, indicating that germ cells were indeed forming. Germ cells were greatly enlarged reaching diameters of up to 45 microns and expressed oocyte-specific markers. In terms of their visual appearance the similarity was striking; both natural oocytes and oocytes derived from stem cells were identical in size and exhibited zona pellucida-like structures. The in vivo germ cell potential of skin-derived stem cells was assessed by mixing them with newborn ovarian cells and transplanting them into the mouse. Dissection revealed that GFP+ cells also appeared in vivo. This study establishes a new model for studying oogenesis and germ cell formation in vitro.

Friday Science Review: May 27, 2011

Functioning Neurons from Canine Embryonic Stem Cells

University of Guelph ♦ University of Toronto ♦ Published in PLoS ONE, May 17, 2011

Scientists have successfully created functioning neural cells from canine embryonic stem cells (ESCs). The protocol used was similar to those used to create neural stem cells from human ESCs. In order to produce neural cells, ESCs were subjected to treatment with epidermal growth factor (EGF) or the signaling molecule Noggin. In both cases, treatment led to the formation of neural stem/progenitor cells expressing the neural lineage marker SOX-2. After priming these neural stem cells, continuing differentiation in the presence of specific growth factors at low dose led to the formation of a diversity of different canine neural cells including oligodendrocytes and astrocytes.

In order to test the functional maturity of the ESC-derived neural cells, researchers grew them along with primary canine fetal cells and astrocytes and then took patch-clamp recordings; a method that can measure action potentials as they fire down the length of the neuron. These recordings showed that the stem cell-derived neurons received inhibitory and excitatory synaptic inputs similar to those observed in functioning neural cells. This protocol provides a necessary proof-of-concept and suggests that the canine could potentially be used as a clinically relevant animal model to improve the quality of data gathered in preclinical studies of stem cell transplantation.

The Protein Localizome

University of British Columbia ♦ Published in PLoS ONE, May 17, 2011

In a particularly eloquent study, researchers have exploited a specific cell type in the nematode worm, C. elegans, in order to begin elucidating the cellular location of proteins. Studies of protein localization are of great value to the scientific community as localization and function are often associated. The large size of the body wall muscle cells in C. elegans and the degree of order exhibited by the sarcomeres within, provide an excellent stage for experimentation and discovery of protein position.

In the beginning of what could become a complete protein “localizome”, researchers unveiled the location of 227 proteins. They achieved this by tagging proteins with green flourescent protein, a molecule that can be observed using flourescence microscopy. The localizations of proteins investigated, orthologs and homologs of human proteins, were largely unknown as no data was available on their precise positioning in the cell. Researchers observed 14 sub-cellular localization patterns in addition to discrete localizations. It is expected that this data will be useful in understanding muscle sarcomere assembly and function, and be applicable in the development of therapeutics for skeletal muscle diseases in humans.

Ultra-Sensitive Detection of Infectious Agents in Human Tissue

Genome Sciences Centre ♦ University of British Columbia ♦ Published in PLoS ONE, May 13, 2011

With the advent of massively parallel sequencing technology it is possible to detect as little as a few RNA transcripts in a sample of human tissue. This technology can analyze an entire transcriptome and pick out the needles in the haystack, so to speak, by identifying low abundance transcripts. In this work researchers describe an approach to troll large sequence data sets for microbial sequences. The impressive part is the sensitivity of the platform. Researchers sequenced RNA libraries containing decreasing quantities of an RNA-virus and found that viral transcripts could be detected at frequencies of less than one in a million. When as much as 20% of cancers are caused by infectious agents, a technology of this nature could have great utility as a diagnostic platform and is certainly more efficient than the traditional culture-based method for identifying infection.

Friday Science Review: May 20, 2011

Genetic Architecture: How a Cell is Wired

University of Toronto ♦ National Institute of Health ♦ Albert Einstein College of Medicine

Published in Nature Biotechnology, May 15, 2011

A great deal of effort has been invested in elucidating the physical interactions of proteins in order to understand their functional relationships. The research community is also trying to reveal the functional connections between genes. Very few genes that display genetic interaction actually physically interact however, so mapping out the architecture of genetic interactions and how these fit into signaling pathways must be done without using physical association. Researchers in Toronto have used a method known as dosage suppression to plot hundreds of genetic interactions in yeast and plotted these together to create a global genetic interaction network map that can be leveraged to understand how the cell is wired on a high level.

Dosage suppression is a form of genetic interaction that occurs when over-production of one gene compensates for another gene that has been silenced due to mutation. In this study gene expression was bolstered through the ectopic expression of yeast genes. In certain cases, this up-regulation can lead to the rescue of a mutant gene, at which point a genetic interaction or ‘edge’ is established. Researchers collected dosage suppression genetic interactions for 424 essential genes that were annotated in the Saccharomyces Genome Database. These interactions gave rise to a network containing 768 genes with 1,293 genetic interactions. The network was visualized using a program known as Cytoscape in order to place genes with common dosage suppression in distinct clusters. A clustering analysis identified nine clusters, each composed of 30 or more genes corresponding to specific biological processes. Researchers further analyzed the network to illustrate how it can be used to provide mechanistic insight into pathways and complexes, and showed that PKA signaling is linked with kinetochore function.

Dosage suppression is relevant to the development of cancer as gene amplification and gain-of-function mutations are associated with up-regulated gene activity, disease initiation, and progression.

Combination Treatment Cures Breast Cancer in 40% of HER-2+ Transgenic Mice

Saskatchewan Cancer Agency ♦ University of Saskatchewan

Published in Cancer Gene Therapy (npg), May 13, 2011

Human epidermal growth factor receptor (HER-2) is over-expressed in roughly 20% of breast cancer incidents. In some cases of HER-2-positive breast cancer, the humanized anti-HER-2 antibody Trastuzumab (Herceptin) is used for therapy. Unfortunately, most patients that receive the antibody develop resistance to it in time. As a result, the research community has been looking for alternative strategies to target HER-2. Researchers in Saskatchewan have recently released some very impressive results from an animal study wherein mice were treated with an adenovirus-based vaccine.

Adenoviruses can be engineered to express any number of transgenic proteins that elicit potent transgene-product specific CD8+ T-cell responses following injection into the body. In this study researchers constructed a HER-2-expressing adenovirus that was capable of inducing a significant reduction in breast carcinogenesis in transgenic mice. It was noted, however, that the vaccination had to be given prior to tumour formation. Mice that had pre-existing tumours did not benefit from the vaccine. Trastuzumab, while being highly effective at stunting the growth of early-stage tumours, has little to no effect on larger, more mature, tumours. After combining Trastuzumab therapy with the recombinant adenovirus vaccine, researchers realized they had stumbled upon a rather effective treatment. Mice, with well-established tumours, receiving combination therapy did much better than those receiving either monotherapy; 4 out of 10 mice treated were entirely cured of the condition, while there was significant delay in death of the remaining 6 tumour-bearing mice.

These findings suggest that a combinatorial immunotherapy of a HER-2 expressing adenovirus and Trastuzumab could be a new therapeutic approach to the treatment of advanced HER-2 positive breast cancer.

Friday Science Review: May 13, 2011

Environmental Stimuli Enhance Visual System Function

McGill University ♦ Published in Neuron (Cell Press), May 12, 2011

The developing nervous system utilizes sensory inputs to lay down the correct neural circuits, strengthening and weakening specific connections where necessary. Sensory cues from the external environment can play a role in neural development as well.  A new study from McGill illustrates that acute environmental stimuli can have strong and long-lasting effects on both synaptic plasticity and functional refinement during development. Using the Xenopus tadpole as a model to investigate the matter, researchers analyzed the effects that 20 minutes of visual stimulation had on the development of the visual system. They found that visual stimulation increased transcription of brain-derived neurotrophic factor (BDNF), a protein that is involved in refining the nervous system by strengthening appropriate synaptic connections and eliminating those that are inappropriate. The effects of visual conditioning went beyond increasing expression of BDNF in the short-term, it also led to improved visual acuity following the completion of development. Animals that were conditioned with visual stimulation and then returned to their normal rearing environment had improved visual system function, as measured by their overall visual acuity.

Array-based Platforms for CNV Analysis: Establishing a Benchmark

Hospital for Sick Children ♦ University of Toronto ♦ Harvard Medical School ♦ Sanger Institute ♦ Uppsala University ♦ University College of Medicine

Published in Nature Biotechnology, May 8, 2011

The largest component of genomic variation within humans lies in copy number variants (CNVs), segments of DNA that duplicate causing expanded regions in the genome. Differences in this variable genetic content between individuals in part explains the differences that humans have in their susceptibility to disease. The detection of CNVs has been playing an ever-more important role in cancer research, clinical diagnostics, and genome-wide association studies. To date, the scientific community has discovered over 15,000 CNVs in the human genome which have been logged in the Database of Genomic Variants. Despite the known importance that CNVs play in disease pathology, there remain several factors related to CNV detection platforms that have hampered the use of this data in research and clinical settings.

The two primary platforms for CNV detection are comparative genomic hybridization arrays and single nucleotide polymorphism arrays, a number of which have been released over the last several years with a trend toward higher resolution. The absence of standardization in CNV reporting and reference samples makes comparing platforms exceedingly difficult, which is exacerbated by a range of platforms that have different genome coverage and resolution. Another complication are the algorithms used to “call”  or identify CNVs, which themselves can be quite different. There has obviously been a need for a robust comparability study between today’s current platforms, and this recent study is the first that establishes a benchmark for these platforms to live up to.  Researchers carried out a comprehensive evaluation of 11 CNV detection platforms, looking at data quality, CNV calling, reproducibility, concordance across array platforms and laboratory sites, amongst other things.

Each array was used to analyze six well-characterized control samples in triplicate. As would be expected newer arrays outperformed older arrays when it came to the number of CNV calls and the reproducibility of calls; likely due to their higher resolution and the performance of their probes. An important finding to drive home is that the choice of analysis tool can be just as important as the choice of microarray for accurate CNV detection. When using identical raw data, different algorithms gave rise to considerably different call numbers of varying quality. Researchers suggest that customized algorithms be made for individual platforms and specific data types to reduce this variation. Overall, this assessment of array-based platforms should stress the importance of experimental design in CNV discovery and association studies, to ensure that the reliability and consistency of CNV detection platforms is upheld for their future use in the clinic. The authors of this work have made all of their raw data available to the scientific community providing an extraordinarily robust reference set for future analysis.


Friday Science Review: May 6, 2011

Heterochromatin Structures Disperse as Somatic Cells Move to Pluripotency

University of Toronto ♦ Hospital for Sick Children ♦ Ontario Human iPS Cell Facility ♦ Sprott Centre for Stem Cell Research

Published in EMBO Journal, May 4, 2011

Cellular reprogramming of adult cells is achieved through the introduction of genetic factors that make widespread changes to the genome. A hallmark of this process is the remodeling of the epigenome which establishes repressive epigenetic marks at specific locations of genetic code. The presence of repressive marks prevents the transcription of DNA, silencing genes, while their absence leaves genes free for transcription and expression at the protein level. Reprogramming occurs in gradual steps, allowing for both partially and fully reprogrammed states. Partially reprogrammed cells are pseudo-stem cells, possessing some of the qualities of true stem cells, but not all. The exact nature of the structural changes that occur during reprogramming are largely unknown.

A recent study used spectroscopic imaging to identify the changes that occur in heterochromatin during remodeling of the epigenome. Researchers found that heterochromatin was densely packed in the centre of the chromosome in somatic cells and partially reprogrammed iPS cells. On the contrary, there were no clearly defined boundaries of heterochromatin in embryonic stem cells or fully reprogrammed cells. Instead, heterochromatin was structured irregularly into 10nm fibres dispersed across the chromosome. To determine whether chromatin reorganization was a characteristic of achieving the fully reprogrammed state researchers brought partially reprogrammed  iPS cells to the fully reprogrammed state using a a cocktail of cell signaling inhibitors. The results showed that reorganization does indeed occur in the latter portion of the reprogramming process.

Enzymatic Modification of Glycoproteins in the Lab

University of Guelph ♦ Published in PNAS, May 3, 2011

Proteins can be powerful therapeutics for the treatment of human disease, however, their use is often hampered by processes within the body that reduce their half-lives and circulation time. Instability, break down by enzymes, neutralization by antibodies, and clearance from the bloodstream are known factors that contribute to reduced half-life. One method that can significantly increase the circulation time of a therapeutic protein, and its resulting effect on the body, is the addition of modifiers that make proteins just a little more rugged. Two such modifiers are polyethylene glycol (PEG), and polysialic acid (PSA).

PSA is biodegradable, and unlike PEG, it is non-immunogenic; the development of antibodies against PEGylated therapeutic proteins has raised some concerns in the scientific community. As a result, PSA is an attractive modifier for future therapies. Although PSA has been successfully added to proteins in previous studies by chemical means, this recent study from the University of Guelph marks the first time PSA has been added to specific sites on therapeutic proteins in vitro, using enzymes.

To accomplish this task researchers utilized two transferase enzymes, a -sialtransferase derived from Campylobacter jejuni and a -polysialtransferase derived from Neisseria meningitidis. In order to prove that the enzymatic approach works, two different human therapeutic proteins were subject to treatment: alpha-1-antitrypsin (A1AT, used to prevent uncontrolled tissue breakdown) and factor IX (used to treat hemophilia B). The experiment was a success and both proteins were modified as expected. Researchers took a closer look at A1AT, and the effect that polysialylation had on its activity and stability; their findings were positive. Polysialylation did not affect A1AT’s in vitro inhibition of human neutrophil elastase. After injecting modified A1AT into mice, its pharmacokinetic profile was significantly improved.

Friday Science Review: April 29, 2011

Gene Transfer Restores Regenerative Power to Circulating Angiogenic Cells

St. Michael’s Hospital ♦ University of Toronto

Published in Molecular Therapy, April 26, 2011

An interesting new approach to autologous cell therapy for patients with coronary artery disease uses gene transfer to enhance the regenerative activity of circulating angiogenic cells (CACs). These rare cells circulate freely throughout human vasculature, and are of interest to researchers because of their ability to promote the formation of new blood vessels and contribute to endogenous vascular repair. The regenerative capacity of these cells is impaired in humans that have cardiovascular disease, however, limiting their use as a therapeutic tool for this patient group.

Noting that the regenerative activity of CACs is linked to the availability of nitric oxide  (NO), researchers at St. Michael’s Hospital and the University of Toronto hypothesized that up-regulation of the gene encoding endothelial NO synthase (eNOS) could remedy the problem. This enzyme, typically found in the vasculature, is responsible for endogenous NO production. To test their theory, researchers used a virus to transduce a construct containing the eNOS gene directly into CACs from patients with coronary artery disease; first they looked at the results in vitro. The modified CACs were able to contribute to angiogenic tube formation when grown in the petri dish along with umbilical vein endothelial cells.

A look at the capabilities of transduced CACs in vivo confirmed the in vitro findings. Transduction of the eNOS gene restored the ability of CACs to improve ischemic hind limb perfusion in an animal model. These data support the notion that CACs may prospectively be used to help repair vasculature in patients with coronary artery disease. In this treatment paradigm, CACs would be removed from patients, isolated in the lab, transduced with the eNOS gene, and then injected back into the patient for tissue regeneration.

Ex Vivo Lung Perfusion Determines Viability of High-Risk Donor Lungs

Toronto Lung Transplant Program ♦ Published in NEJM, April 14, 2011

A large majority of donor lung transplants fail to meet the criteria for lung transplantation. A combination of the damage that occurs following brain death, and the injury that occurs to lung tissue in the ICU, renders them unsuitable for medical use. When using sub-optimal donor lungs, graft dysfunction is prevalent, leading to acute lung injury that occurs within the 72 hours following transplantation.

Normothermic ex vivo lung perfusion (EVLP) was developed by a group of researchers in Toronto to increase the percentage of donor lungs that can be used for transplantation. This biotechnology allows for the perfusion of lungs at body temperature in an ex vivo circuit that attempts to mimic normal physiological conditions. EVLP lasts for roughly 4 hours, and provides a means to quantitatively assess the function of donor lungs prior to transplantation. In this clinical study, funded by Vitrolife, researchers assessed the feasability of transplanting high-risk donor lungs into patients following EVLP.

During the clinical study a total of 136 donor lungs were transplanted. Of these, 23 were classified as high-risk and underwent EVLP prior to transplantation, while the remainder made up the control group. The primary endpoint of the study was graft dysfunction after 72 hours; secondary endpoints included 30-day mortality, bronchial complications, duration of mechanical ventilation, and length of stay in the hospital ICU. No significant differences were observed in any of the secondary endpoints between treatments. The incidence of graft dysfunction in patients who received high-risk donor lungs prepared with EVLP was only half of that observed in patients receiving donor lungs considered to be “standard” by clinicians (15% and 30% respectively). These results indicate that EVLP does a better job selecting donor lungs for transplantation.

Friday Science Review: April 22, 2011

Fibroblast Growth Factor 9 Helps Form Vasoreactive Vessels

University of Western Ontario ♦ Published in Nature Biotechnology, April 17, 2011

Some interesting findings from the University of Western Ontario could have implications for future angiogenesis therapies and tissue engineering approaches to the treatment of vascular disease. Researchers discovered that fibroblast growth factor 9 (FGF9), an angiogenic growth factor, contributes to the development of vasoreactive blood vessels. Two primary processes must occur within implants to produce functional grafts. Firstly, endothelial cells must be stimulated to produce angiogenic sprouts, and secondly, these sprouts must be muscularized by being wrapped in smooth muscle cells. The latter of these two processes is crucial for vasoreactivity — the ability of vessels to alter the luminal diameter to control the flow of blood into capillary beds. Fibroblast growth factor 2 (FGF2)  has been shown to stimulate endothelial cells to form angiogenic sprouts, but its utility in producing functioning grafts has been limited because it does not stimulate the formation of cords of smooth muscle cells that allow for vasoreactivity. FGF9, on the other hand, fails to produce angiogenic sprouts but seems to direct mesenchymal cells to produce the muscle necessary for control over luminal diameter. Researchers show that delivering FGF9 to implants produces stable, durable, and vasoresponsive blood vessels that can remain physiologically competent for at least a year. This research challenges the notion that endothelial cells must be targeted for vascular repair, and suggests that targeting mesenchymal cells may be the more crucial consideration in developing angiogenesis therapies. It will likely be a combination of targeting both cell types and using a variety of angiogenic growth factors, including FGF2 and FGF9.

B Cells Drive Insulin Resistance in Animal Model

Stanford University ♦ University of Toronto ♦ Duke University Medical Center

Published in Nature Medicine, April 17, 2011

One of the key drivers of insulin resistance and glucose intolerance is chronic inflammation of the visceral adipose tissue (VAT). Inflammation of VAT is caused by the infiltration of macrophages that produce proinflammatory cytokines, and infiltration by T cells that also trigger inflammatory mechanisms. It now looks like B cells are in the mix as well, exhibiting a pathogenic role in the development of metabolic abnormalities. Using a knockout mouse model that fails to produce mature B cells, researchers showed that mice without B cells had lower fasting glucose and greater glucose tolerance than wild type mice. Knockout mice also had reduced fasting insulin and improved insulin sensitivity, and fewer proinflammatory macrophages in VAT compared to normal animals. Investigation into the mechanisms in which B cells promote metabolic abnormality led to the finding that B cells activate T cells through the presentation of an MHC complex, and that this was linked to glucose tolerance. In addition to modulating T cells, B cells also release IgG antibodies that regulate immune function. It was found that IgG in VAT induced a considerable decline in glucose tolerance, the effects of which were associated with decreased fasting insulin, one of the hallmarks of insulin resistance. Treating mice with CD20, a B cell-depleting antibody, attenuated disease, while transfer of IgG antibodies from mice with diet-induced obesity to normal mice rapidly induced glucose intolerance.

Friday Science Review: April 15, 2011

New Prognostic Signature for NSCLC

The Campbell Family Cancer Research Institute ♦ University of Toronto

Published in PNAS, April 7, 2011

It has long been known that the tumour microenvironment, or niche, plays a major role in the development of cancer, the progression of disease, and eventually metastasis. Non-small cell lung carcinoma (NSCLC), which accounts for 85% of lung-cancer related deaths, is highly prone to influence from fibroblasts in the stroma surrounding cancer cells.  Researchers at The Campbell Family Cancer Research Institute have been investigating one of the leading causes of cancer cell invasion, a process known as desmoplasia. During desmoplasia several things occur that exacerbate disease conditions, one of which is the evolution of normal fibroblasts to what are known as carcinoma-associated fibroblasts (CAFs). It seems that CAFs are able to enhance the tumorigenicity of lung cancer cell lines, and have a degree of prognostic power in assessing outcome. Using 15 resected NSCLCs, Dr. Tsao and his team established 15 matched normal fibroblast and CAF cell lines and carried out microarray gene-expression analysis to identify differentially expressed genes. In doing so the lab group identified 46 genes that were either upregulated or downregulated between the two sets of cells. A subset of 11 genes from this group was able to form a prognostic gene expression signature that was subsequently validated in several NSCLC microarray datasets. It was found that these genes encode extracellular matrix proteins that are regulated by the TGF-β signaling pathway. Protein-protein interaction analyses suggested that the focal adhesion and MAPK signaling pathways are involved in the transition from the normal to carcinoma-associated fibroblast states.

Bioinformatics Approach Reveals Resistance-based Genes

Mount Sinai Hospital ♦ Translational Genomics Research Institute (TGen), Arizona

Published in PLoS ONE, April 4, 2011

Statins have traditionally been used to lower cholesterol but recent evidence suggests they may have a future in treating cancer. Cholesterol accumulates in cell membranes in structures known as lipid rafts, which can influence processes such as cell growth and survival. By augmenting the composition of these lipid rafts, statins may exert cytotoxic effects on cancer cells. Many cancer cell lines exhibit resistance to statins though, limiting their utility as cancer therapeutics. In this eloquent study researchers used a panel of cancer cell lines (NCI60) and publicly available genomic and pharmacological data, to identify genes that conferred resistance to two statins, simvastatin and lovastatin. Pharmacological data identified simvastatin and lovastatin resistant cell lines which were then analyzed by whole-genome single marker association tests to uncover genes that regulate resistance. Three genes (NRP1, COL13A1, and MRPS31) were linked to resistance to simvastatin while another six (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) were linked to resistance in the case of lovastatin. To confirm their findings researchers carried out a functional validation with the gene EAF2. Silencing this gene with RNAi changed the response of the colon cancer cell line HCT-116 to both statins. This work really illustrates the power of today’s genomic and pharmacological databases, and how they can be leveraged to provide insight into treating cancer.

Friday Science Review: April 8, 2011

Cancer Immunotherapy in the Clinic: Dendritic Cells Present the Possibility

McMaster University ♦ Medical School of the Vrije Universiteit Brussel

Review Published in Molecular Therapy (npg), April 5, 2011

Dendritic cells play a vital role in the generation of T-cell responses to invading pathogens in the body. They fall into a class of cells known as antigen presenting cells (APCs), that display small protein segments, otherwise known as antigens, to T-cells such that an immune response may be mounted against foreign pathogens. There are three mechanisms of activation that lead to a rapid and efficient immune response. The first is the recognition of an antigen on the surface of APCs by T-cells. After a T-cell and APC meet one another, the second signal occurs by way of reinforcement through the interaction of costimulatory ligands on the extremity of both cells that work to enhance the first signal and ensure the T-cell acts upon its recognition of the foreign antigen. The third signal comes in the form of cytokines, small soluble chemical messengers released by APCs that promote T-cell polarization — immune activity geared towards foreign invaders. Dendritic cells are unique in their ability to trigger all three of these signaling mechanisms, and thus the proposition of using this cell type in immunotherapy vaccines is an interesting one.

Without knowing it, scientists have been moving towards utilizing immunotherapy in the clinic for more than 100 years. In the early 20th century researchers were already clueing in to the fact that there may exist some form of immune surveillance system allowing specific cell types to recognize and eradicate transformed cells from the body. In this recent review, written in part by researchers at McMaster University, the topic of immunotherapy is discussed in the context of manipulating dendritic cells to be amenable for use in immunotherapy vaccines.  The general concept is to engineer dendritic cells such that they express small peptides known as tumour-associated antigens (TAAs). After being injected into the body TAAs elicit an immune response polarized towards tumour cells in the body. This is achieved with genetic manipulation in the petri dish prior to preparing the vaccination. During this process a single gene or multiple genes can be introduced to dendritic cells, resulting in the continuous production of native TAA peptides that are packaged and delivered to the cell surface on MHC class II molecules.

Each of the three signaling mechanisms that promote T-cell response may be exploited for immunotherapy in vivo. The foundation of this therapeutic approach to cancer is T-cell recognition of the tumour-specific antigen on the surface of engineered dendritic cells. This recognition is translated to action by receptor/ligand costimulation, which can be magnified by one of two means: enhancing the expression of costimulatory molecules, or downregulating molecules that inhibit or suppress T-cell response to dendritic cells. This is achieved by gene transfer or silencing mechanisms in vitro. Enhancing the CD40-CD40L receptor/ligand pair and downregulating the inhibitory zinc-finger protein A20 have proven effective in increasing overall costimulation at the T-cell/APC interface. Modifying the cytokine and chemokine milieu, the environment in which the immune cells reside, helps to direct the polarization of the ensuing immune response. Anticancer T-cell responses are best suited to occur in polarized niches established by type I IFN, IFN-γ, and IL-12p70. These niches are characterized by the presence of CD8+ T-cells (cytotoxic T-cells), CD4+ T-cells (helper T-cells), and natural killer cells. To achieve modification of the immune microenvironment dendritic cells are engineered to continuously express various cytokines and chemokines.

So where do we stand in the clinic? Protocols for the generation of dendritic cells from monocytes have been established and implemented to create immunotherapies for the clinic. Phase 1 studies illustrate that immunotherapy is well-tolerated. The preparation of dendritic cells for immunotherapy applications is somewhat of a recipe. Dendritic cells are first mixed with tumour-associated antigens, and then matured with the addition of a cocktail of cytokines including PDE-2, IL-β, IL-6, and TNF-α. Improvements upon this protocol using IFN I produce “DC1″ dendritic cells that create a more highly polarized immune response following administration. One dendritic-cell based cellular vaccine has been approved by the FDA for the treatment of prostate cancer. This immunotherapy, Sipuleucel-T, was used to treat patients with castration-resistant prostate cancer and was reported to extend patient survival by 4.1 months. The authors of this review completed a clinical study involving 35 patients with metastatic melanoma. Roughly 60% of patients under therapy mounted an immune response against one or more of the vaccine antigens. Disease control of greater than 6 months with regression of metastases was noted in 35% of patients, while recurrence free survival was 23 months.

Things are looking promising in this emerging field with demonstrable safety and several clinical studies underway. While T-cell activation appears to be consistent, current engineered dendritic cells fail to provide strong enough costimulation to maintain a proinflammatory immune environment and recruit all of the necessary components necessary to eradicate transformed cells. As tumours maintain immunosuppressive environments successful immunotherapies will have to elicit persistent and aggressive responses to tumours in vivo. This will likely be achieved through the transfer of several genetic components that simultaneously enhance all three mechanisms of immune response activation.

Friday Science Review: April 1st, 2011

Temperature Sensitive Yeast Library Poised to Uncover Gene Function

University of Toronto ♦ Published in Nature Biotechnology, Mar. 27, 2011

In efforts to document the roles of essential eukaryotic genes, a group of researchers at The Terrence Donnelly Centre for Cellular and Biomolecular Research have constructed an expansive library of yeast mutants that can be manipulated with temperature to provide insight into gene function. A set of 787 temperature-sensitive strains was produced by amplifying temperature-sensitive alleles from yeast mutants and then integrating these back into a common genetic background at their native loci. To allow for the use of high-throughput screening methods a selectable marker (kanMX) was linked to each allele.

Temperature sensitive alleles allow for fine tuning of gene function with three conditions or states: permissive, semi-permissive, and restrictive. In this study, led by Dr. Charles Boone, chemical-genetic suppression analyses were carried out on the temperature-sensitive mutant collection using small molecule compounds at both permissive and non-permissive states. If a yeast mutant grows under non-permissive conditions, it can be concluded that it contains a mutation that is suppressed by a given small molecule. By creating a library populated with mutant strains with a common genetic background, researchers were able to use high-throughput strain manipulation and then carry out high-content screens at the single cell level. Gene function was determined by visualization and quantitative measurement of specific morphological features. In order to score the entire library Dr. Boone’s group created customized software that carried out automated image analysis using features such as cell shape, budding index, organelle density and a panel of 85 more reporter-specific parameters. Impressively, all computationally derived phenotypes were confirmed manually with no discrepancy in findings. Researchers took the library for a test run and performed quantitative analysis of a GFP-tubulin marker revealing that condensin and cohesin have roles in spindle disassembly.

Researchers believe the mutant library will be amenable to exploration with high-throughput methods such as high-resolution growth profiling, chemical-genetic suppression, and high content screening to elucidate the role of highly conserved signaling pathways in the model organism. Yeast has 1,101 essential genes; the mutant collection created here represents 45% of these (497 alleles). A non-overlapping set of mutants created recently covers another 250 alleles. Taken together these collections cover roughly 65% of the essential genes in yeast and are a powerful means to begin annotating the functions of all of yeast’s essential genes.

Normalizing Src-Kinase Enhancement of NMDAR, A New Paradigm for Treating Schizophrenia

The Hospital for Sick Children ♦ University of Toronto ♦ Tufts University School of Medicine

Published in Nature Medicine, Mar. 27, 2011

Excessive NRG1β-ErbB4 signaling in the brain is a hallmark of individuals suffering from schizophrenia. This aberrant signaling is believed to contribute to the hypofunction of a specific glutamate receptor, NMDAR, that is crucial for synaptic plasticity and long-term potentiation at Schaffer collateral-CA1 synapses in the hippocampus and prefrontal cortex. A popular hypothesis for the cognitive deficits and hallucinations associated with schizophrenia has been that they are the result of the general hypofunction of NMDAR.

Dr. Michael Salter and his team at The Hospital for Sick Children proposed something a little different. They hypothesized that the underlying cause of schizophrenic symptoms was actually the result of interference in a cellular mechanism that enhances NMDAR function. The tyrosine kinase Src is the primary agent mediating NMDAR phosphorylation and enhancement, and is also involved in promoting NMDAR-dependent long-term potentiation. For these reasons Dr. Salter and his team sought to investigate whether NRG1β-ErbB4 signaling has an effect on Src-mediated phosphorylation of NMDAR. It turns out it does.

Analysis of whole-cell recordings of neurons in the CA1 layer of acute slices of hippocampus removed from adult animals revealed that NRG1β-ErbB4 signaling does indeed affect Src-mediated enhancement of NMDAR function. The same was found in slices of prefrontal cortex. Researchers believe that NRB1β signaling, via its cognate receptor ErbB4, compromises the catalytic activity of Src kinase which in turn interferes with downstream events that require Src-mediated enhancement, including long-term potentiation at Schaffer collateral synapses. NRB1β-ErbB4 signaling could exert its effects by suppressing activators or facilitating inhibitors of kinase function. The work by Dr. Salter and his team provides a vital piece to the mysterious puzzle that is schizophrenia, and identifies a novel therapeutic regime to tackle cognitive dysfunction associated with the disorder.

Friday Science Review: March 25, 2011

Beware of Repeats

The Hospital for Sick Children ♦ University of Toronto ♦ Published in PLoS Genetics, Mar. 10, 2011

Trinucleotide repeats are known to be associated with the onset of many diseases including Huntington’s disease and fragile X syndrome. These unstable elements can be transcribed bidirectionally and are dynamic, meaning their numbers can change within individuals and across generations. Particularly worrisome elements include CAG and CTG repeats. In this recent review, Dr. Christopher Pearson describes a process known as Repeat Associated Non-ATG translation (RAN-translation) wherein portions of DNA containing blocks of CAG repeats can be transcribed in the absence of a conventional ATG transcriptional start site. 

Repetitive tracts of DNA can be transcribed in all three reading frames giving rise to RNA transcripts that produce polymeric proteins composed of repeating amino acid building blocks. Unstable repetitive genetic elements may be toxic to the body in several ways, leading to loss-of-protein expression, over-expression of normal proteins, and toxic gain-of-protein function. Due to the functionality of proteins, diseases are often physically manifested at the protein level. Interestingly, when it comes to diseases caused by unstable trinucleotide repeats, RNA transcripts can get in the mix aswell. Transcripts containing repeating CUG or CGG elements can have toxic gain-of-function effects. It is even possible for diseases to be caused through the combined action of a toxic gain-of-function RNA and related toxic polymeric protein.

Acetylcholine, Turning Down the Action

University of Western Ontario ♦ Published in PLoS ONE, Mar. 10, 2011

It has been difficult to assess the contribution that acetylcholine (ACh) has on locomotion. As a major peripheral neurotransmitter it has a vital role in controlling movement, emotional behaviour, and is also involved in memory and learning. Dysfunction in cholinergic activity is involved in the development and onset of many different disorders of the brain, including, but not limited to, Alzheimer’s, schizophrenia, Parkinson’s disease, epilepsy and ADHD. The first attempts at recreating cholinergic dysfunction used non-selective means that were either too destructive, causing destruction of neural types beyond cholinergic, or not destructive enough, failing to eradicate all cholinergic neurons. This short coming left room for significant variation in previous studies, making it difficult to elucidate the effects that ACh has on the various nervous systems.

The alternative to mimicking cholinergic dysfunction was to approach the situation from the standpoint of genetics. This is what Dr. Vania Prado and her lab team have been working on in recent times. This was no easy feat though. To impair cholinergic signaling they chose to target the vesicular acetylcholine transporter (VAChT), a protein that is involved in sequestering acetylcholine and accumulating it within vesicles for transport. The VAChT gene, however, is located within the first intron of the acetylcholine transferase (ChAT) gene in a nested formation. In order to knock down expression of VAChT they flanked it with short “lox” sequences that are used to remove the gene.

What the group found was that the new mutant mice strains they had generated exhibited hyperactivity when exposed to new environments. A similar trait is observed in patients suffering from Alzheimer’s, schizophrenia, and ADHD. Rescuing VAChT expression alleviated that hyperactive phenotype suggesting that acetycholine serves the purpose of toning down the nervous system to control locomotion and other peripheral body functions.

Friday Science Review: March 18, 2011

Alum Explained

University of Calgary ♦ Published in Nature Medicine, Mar. 13, 2011

During the administration of a vaccine, an antigen is delivered along with another substance, known as an adjuvant, which arouses the immune system and increases overall effectiveness. The most common adjuvant in use today is alum, a trivalent aluminum-containing salt in crystal form.

Many questions related to alum’s mechanisms of action remain unanswered, or were mostly unanswered until the emergence of recent findings from the lab of Dr. Yan Shi at the University of Calgary. Dr. Shi and his lab group discovered that alum interacts with dendritic cells, a specific cell-type in the immune system that specializes in digesting antigenic material and presenting it on the cell surface. There is no specific receptor for alum however, instead it interacts with lipids on the plasma membrane eliciting a lipid sorting mechanism. Sorting of lipids induces a phagocytic response causing an influx of antigen into dendritic cells and an increase in affinity for CD4+ T cells.

Alum has massive implications for human health given the size and importance of the vaccine market. This new insight into dendritic cell response to alum will likely be leveraged to improve upon the efficacy of future vaccines.

Suicide Gene Delivers the Blow

Jewish General Hospital ♦ McGill University ♦ Published in Cancer Gene Therapy, Mar.11, 2011

Combination treatment paradigms for cancer have been under investigation for some time, but the suicide gene approach outlined in this recent research exemplifies the advances that have been made in the area. In this approach a tumour-specific oncolytic virus delivering a fusion construct is paired with a non-toxic prodrug. When the prodrug enters an infected cell containing the suicide transgene it is broken down by the cell’s machinery into toxic metabolites. In essence, the cell commits suicide.

Oncolytic viruses target cancer cells by taking advantage of their genetic abnormalities. A perfect example is vesicular stomatitis virus (VSV), a single stranded RNA virus that grows like wildfire in cancer cells but is unable to populate healthy cells. How? VSV is extraordinarily sensitive to type-1 interferon mediated immune responses. In normal cells that have interferon signaling cascades intact the virus cannot replicate. However, in cancer cells, which have genetic alterations affecting the interferon pathway, the virus survives with relative ease. Researchers utilized a suicide gene (CD::UPRT) in combination with 5-FC, a non-toxic prodrug that is metabolized to the toxic 5-flourocytosine (5-FU) form in the presence of cytosine deaminase. The deamination of 5-FC leads to its conversation to 5-FU, a small molecule drug commonly used in chemotherapeutic regimes for the eradication of cancer. The introduction of 5-FU into the cellular system prevents normal DNA replication, and hence causes cell cycle arrest. In this study researchers showed that the suicide gene strategy was able to trigger oncolysis in a number of VSV-resistant cell strains, including prostate PC3, breast MCF7, B-lymphoma Karpas, and melanoma B16-F10.

The combination scheme investigated here allows for the targeted removal of tumour cells while preserving healthy cells. As such, it circumvents one of the primary barriers associated with the development of efficacious cancer therapeutics − non-selective toxicity. 5-FU is also highly soluble, which causes detriment to neighbouring tumour cells that have been weakly infected, producing a particularly powerful treatment.

Friday Science Review: March 11, 2011

Insulin + Pancreatic Stem Cells, Proof of Life

University of Toronto ♦ Published in Cell Stem Cell, Mar. 4, 2011

The origin of insulin-producing pancreatic β-cells has been a matter of contentious debate. Some research groups have produced findings that would suggest β-cells duplicate themselves and that new β-cells do not arise from the differentiation of a more primitive pancreatic progenitor. Other groups have proven the existence of pancreas-derived multipotent progenitors (PMPs) that are capable of giving rise to a spectrum of cells from both the pancreatic and neural lineage. So where do β-cells come from?

New results from the lab of Dr. Derek van der Kooy point in the direction of PMPs. Lineage labeling experiments in mice showed that PMPs originate from the embryonic pancreas, as opposed to the neural crest, which has often been cited as the source of pancreatic progenitors. These findings are in conflict with previous studies which could not provide evidence of pancreatic progenitors in the developing embryo or the adult. Convincingly, Dr. van der Kooy’s group was able to show that PMPs express insulin in vivo, an attribute that has often been considered prerequisite for the production of β-cells.

Analysis of human islet tissue showed that PMPs also exist in humans, and, similar to the mouse PMPs under observation, were capable of differentiation to both the pancreatic and neural lineages. Both mouse and human PMPs were able to alleviate diabetic conditions in mice and may provide another avenue to explore in the pursuit of therapeutic cells for transplantation therapy.

Selective Pressures Shape the Genomic Integrity of Human iPS Cells During Reprogramming

Samuel Lunenfeld Research Institute ♦ Ontario Institute for Cancer Research ♦ The Hospital for Sick Children ♦ University of Toronto

Published in Nature, Mar. 3, 2011

Coercing fibroblasts to revert to an embryonic stem cell-like state places a good deal of stress on the genome. The consequences of reprogramming can affect the development of safe populations of cells for therapeutics, which is why researchers at the Samuel Lunenfeld Research Institute have been interested in understanding how the reprogramming process affects genomic integrity. The integrity of a genome can be measured using copy number variation (CNV) within a population of cells. CNVs arise from deletions or duplications in DNA; as variation increases, the integrity of the genome declines.

In this study supervised by Dr. Andras Nagy and Dr. Timo Otonkoski, researchers characterized the CNV content of 22 human iPS cell lines and 17 human ES cell lines using Affymetrix SNP arrays. Induced pluripotent cell lines were created by way of retroviral transduction or with the use of a transposon known as piggybac. The number of CNVs in iPS cells was roughly twice that found in ES cells on average and many CNVs found in iPS cells were undetectable in ES cells suggesting that CNVs are generated during the reprogramming process. To the surprise of the group the number of CNVs in iPS cells was greatest at early stages, and decreased as cells were passaged in culture.

Researchers hypothesized that the reduction in copy number variation could be the result of two things, firstly, a DNA repair mechanism that corrects deletions and additions as the cells grow and divide in vitro, or mosaicism in early cultures followed by selection of iPS cells that have lower variation and greater genomic stability; a survival of the fittest in a way. It is unlikely that a DNA repair mechanism could operate fast enough to account for the rapid reduction of CNVs observed in iPS cells in culture and indeed, after using fluorescence in situ hybridization (FISH), it was confirmed by the lab group that mosaicism did exist in early cultures of iPS cells. In order to prove that selection was driving the decrease in CNVs researchers focused on deletions that cannot be corrected by DNA repair mechanisms. They found that several of these deletions were selected against during passaging of iPS cell lines. This pressure was bidirectional however, as some CNVs were selected for, not against.

CIITA, A Promiscuous Partner in Lymphoid Cancers

Centre for Translational and Applied Genomics ♦ BC Cancer Agency ♦ University of British Columbia

Published in Nature, Mar. 2, 2011

Chromosomal translocation events are a common abnormality leading to the development of cancer but few have been described as contributors to the development of lymphoid cancers. A new chromosomal translocation event has been implicated in the development of Hodgkin lymphoma and primary mediastinal B-cell lymphoma (PMBCL). Large scale mutations of this nature occur when non-homologous chromosomes transiently stick together and cause breakages that lead to the exchange of genetic information. If genes are placed next to one another following the translocation event, fusion transcripts are created which can lead to cellular abnormalities and malignant expansion.

Genome-wide mapping of translocation events can be carried out using paired-end sequencing of expressed transcripts. Researchers used such a platform to analyze two Hodgkin lymphoma cell lines. Analysis uncovered a highly expressed gene fusion between the MHC class II complex (CIITA) and an uncharacterized gene. Further analysis of 263 B-cell lymphoma cell lines went on to show that the CIITA translocation was highly recurrent in PMBCL (38%) and Hodgkin lymphoma (15%). The genetic event appears to be quite specific to PMBCL as it was observed in only 3% of diffuse large B-cell lymphoma cell lines.

Researchers note that although certain translocation events of very specific rearrangements are key contributors to some B-cell lymphomas, resulting in unique clinopathological features, many well characterized B-cell lymphomas still lack identifiable translocations that define the disease. Translocations in B-cell lymphomas are a rare occurrence, and until the publication of this research, no translocations had ever been reported in PMBCL.

Friday Science Review: March 4, 2011

The Origin of Meier-Gorlin Syndrome

Dalhousie University ♦ University of Montreal ♦ University of British Columbia

Published in Nature Genetics, Feb. 27, 2011

Researchers have mapped a locus for Meier-Gorlin syndrome (MGS), a rare genetic condition characterized by short stature, small ears, and reduced or absent kneecaps. A mutation in the ORC4 gene seems to be at the root of the disorder. ORC4 is a component of the eukaryotic origin recognition complex.

To map the locus responsible for MGS researchers performed high density genome-wide SNP genotyping using a panel of 600,000 markers provided by Illumina. The next stop involved PLINK, a whole genome analysis toolset, which was able to identify a haplotype on chromosome 2 within a number of affected individuals. Sequencing of coding exons located in the ORC4 gene led to the identification of a missense mutation that causes a tyrosine (residue 174) to cysteine switch in the ORC4 protein. The tyrosine residue affected in MGS is completely conserved across eukaryotes suggesting it has an important function; the amino acid is also believed to interact with a conserved arginine residue on a nearby helix motif in the protein structure. In the absence of this interaction the structural integrity of the protein could be compromised in part.

The origin recognition complex consists of six proteins in humans and is essential for DNA replication. It plays a critical role in recognizing origin sites on DNA and in the formation of DNA replication forks. This is the first report of an inherited mutation in any gene of the origin recognition complex in the vertebrate literature.

The Human Serum Metabolome

University of Alberta ♦ National Institute of Nanotechnology

Published in PLoS ONE, Feb. 16, 2011

Human biofluids are very important from a clinical standpoint given the insight they can provide into the disease conditions of a human being. The study of metabolomics attempts to identify, on a large scale, the composition of metabolites found in these biofluids. The advent of advanced analytical techniques along with mounting pressures for scientists in the metabolomics community to document the entire human metabolome, led to the development of the Human Metabolome Project. The project is supported by Genome Alberta and Genome Canada, the latter of which is a private, non-profit, corporation that received $600 million in funding from the Canadian government to develop and implement a national strategy in genomics and proteomics.

The most recent contribution to the project is a comprehensive multicentre study led by Dr. David Wishart at the University of Alberta. Using a diversity of metabolomics platforms researchers were able to identify, and quantify, metabolites found in human serum. The use of different methods, including nuclear magnetic resonance (NMR), and various mass-spectrometry platforms (GC-MS, LC-MS), increased the overall coverage of the serum metabolome. Data gathered via these platforms was linked to computer-aided literature mining which allowed for the development of a virtually complete set of metabolites. In total the group found 4,229 metabolites, but this number may increase in coming years as more powerful characterization techniques are developed.

Dr. Wishart and his colleagues previously characterized the human cerebrospinal fluid metabolome.


Friday Science Review: February 25, 2011

Fusion Construct Promotes Erythropoietic Development from Human Embryonic Stem Cells

McMaster University ♦ The Ottawa Hospital ♦ British Columbia Cancer Agency

Published in Stem Cells, Feb. 15, 2011

The homeobox (Hox) genes encode a group of highly conserved transcription factors that have been known to regulate hematopoietic differentiation. As a result of their involvement in hematopoietic proliferation and lineage commitment, Hox genes have also been implicated in leukemogenesis. Researchers from Dr. Mick Bhatia’s lab have created a fusion construct that alters the hematopoietic differentiation program of human embryonic stem cells (hESCs). They chose the homeobox gene HOXA10, and partnered this with NUP98, a gene that is often found fused to Hox genes in human leukemias. After introduction of the fusion construct into undifferentiated hESCs or early-stage blood progenitors there was a marked increase in the level of erythroid progenitors founds in culture. Introduction of the construct to later-stage cells already committed to the hematopoietic lineage had no effect on the yield of erythropoietic cells. Apparently, unlike some of the fusion constructs observed in leukemias, the combination of HOXA10 and NUP98 does not lead to malignant expansion. Given the interest Dr. Bhatia and his group have in the production of blood cells, it is foreseeable that this fusion construct could be used in future differentiation protocols to bias the differentiation of hESCs towards the erythropoietic lineage.

Ubiquitination Factor E4B, A Novel Target for Brain Cancer

University of Alberta ♦ Published in Nature Medicine, Feb. 13, 2011

The tumour supressor gene TP53 and its protein product p53 are at the root of many cancers. TP53 is inactivated in 50% of human tumours. The resulting deficiency in p53 allows fledgling cancer cells to circumvent apoptotic programs and proliferate wildly. Deficiency in p53 protein may result from a mutation in the TP53 gene, or as is more frequently the case, inactivation of the p53 protein. Inactivation is often the result of ubiquitination. A specific class of enzymes in the body has the ability to add ubiquitin to proteins which marks them for degradation by other protein machinery. However, the molecular mechanisms behind inactivation of p53 in the brain remain largely understood.  Dr. Roger Leng and his team at the University of Alberta have identified a novel mechanism of inactivation in brain tumours: a ubiquitination factor (UBE4B) that directly interacts with the p53 protein destabilizing it and marking it for destruction. The factor was found to interact with an enzyme (Hdm2) that is also involved in ubiquitination. Silencing UBE4B in xenotransplanted tumours led to impaired tumour growth while over-expression of the factor was associated with amplification of its gene suggestive of a positive feedback mechanism. This discovery elucidates a potential target for treating medulloblastoma and ependymoma, two brain cancer types that exhibit inactivation of the p53 protein.

Friday Science Review: February 18, 2011

Mapping the Development of the Pancreatic Lineage

McEwen Centre for Regenerative Medicine ♦ Published in Development, Mar. 2011 (Epub ahead of print)

Human pluripotent stem cells (PSCs) are being investigated as a means to produce insulin-positive cells for the treatment of diabetes. The most efficient mode of producing functional cell types in vitro is to navigate the signaling pathways and temporal cues that lead to their formation during embryonic development. In the case of insulin-producing cells the key is to recreate the pivotal steps in pancreatic development including the induction of definite endoderm, specification of endoderm to the pancreatic fate, and finally the generation of mature endocrine/exocrine cells. Despite the scientific community having a grasp on pancreatic development, current differentiation protocols suffer from low efficiency and an inability to produce homogenous results across a variety of PSC lines using identical treatments. We have yet to identify the optimal signaling pathways that must be leveraged to produce insulin+ cells. Robust differentiation protocols are also hampered by variations in the characteristics of PSC lines which lead to variability in the quality of differentiation cultures. Dr. Gordon Keller and his lab team ambitiously probed this issue by mapping the pancreatic development of several different PSC lines in order to identify the optimal signaling pathways and temporal requirements essential for producing cells of the pancreatic fate.

Keller’s team found that temporally modulating activin/nodal signaling early in the differentiation protocol was crucial for the development of definite endoderm and ultimately for pancreatic differentiation. Wnt signaling and inhibition of BMP signaling at various stages was also prerequisite for the production of insulin+ cells, noting that the degree of BMP inhibition required for efficient differentiation varied extensively amongst PSC lines. By implementing this stage-specific optimization approach for different cell lines, Keller and his colleagues were able to increase insulin expression in cell cultures by a whopping 250 times; some populations contained as much as 25% C-peptide+ cells (prior to C-peptide being cleaved from the pro-insulin molecule, it acts as a linker between the A and B chains of insulin).

This is the second, recent, body of research from Gordon Keller’s lab that emphasizes the importance of identifying the crucial temporal steps that must be satisfied for highly efficient differentiation to terminal cell fates. This paper also reminds us that individual PSC lines will likely require unique treatments in culture to produce maximal results for transplantation therapy.

IL-7 Therapy: A Stimulus Package for the Immune System

Campbell Family Institute for Cancer Research ♦ Published in Cell, Feb. 18, 2011 (Epub ahead of print)

After the immune system succumbs to uncontrollable viral turnover, it eventually fails, leaving the host prone to any number of opportunistic infections. This is the case with HIV infection. One of the primary focuses of HIV research today is the modulation of immune response to encourage the clearance of chronic viral infections. It appears that a certain cytokine, interleukin-7 (IL-7), may be able to prop up the immune system allowing it to move around mechanisms that circumvent immune response during times of chronic infection. In this study led by Dr. Tak Mak, researchers hypothesized that cytokines supporting homeostatic proliferation would be promising candidates for promoting immune response. Indeed, IL-7 did just that.

After administering IL-7 to mice that were chronically infected with lymphocytic chorimeningitis virus (LCMV) variant clone 13, researchers observed an increase in size of the naive T-cell pool, and an enhanced function and cytokine output in LCMV-specific T-cells. IL-7 therapy resulted in clearance of LCMV from chronically infected mice. The cytokine also appears to serve a second function — bolstering levels of the cytoprotective cytokine IL-22. An increase in IL-22 levels has the added benefit of protecting the liver from viral infection, an organ that is particularly prone to damage under the circumstances. Researchers believe IL-7 exerts its effects by downregulating suppressor of cytokine signaling 3 (Socs3) expression in T-cells through the suppression of the FoxO transcription factors.

Friday Science Review: February 11, 2011

Cardiac Differentiation: A Customized Approach

McEwen Centre for Regenerative Medicine ♦ University of Toronto ♦ SickKids

Published in Cell Stem Cell, Feb. 4, 2011

Dr. Gordon Keller of the McEwen Centre for Regenerative Medicine has been a pioneer in the stem cell world and was the first researcher to produce functioning cardiomyocytes from embyronic stem cells. These cells form clusters in the petri-dish that beat in unison, a rather marvelous sight to behold. The efficient differentiation of embryonic stem cells to cardiac cells requires monitoring the very earliest stages of their development. Monitoring the expression of one gene, Flk-1, has been instrumental in recognizing the formation of cardiac mesoderm, an early step in the developmental path of cardiomyocytes. A problem that remains however, is that Flk-1 is expressed in different forms of mesoderm, not all of which lead to the cardiac lineage. A second gene, PdgfR-α, can be used to separate cardiac and hematopoietic lineages when monitored in conjunction with Flk-1. Fractions of differentiating cells that coexpress the two genes have greater cardiomyocyte potential. Keller’s lab group used these two genes to study the stage-specific effects that Activin/Nodal and BMP signaling have on the development of cardiomyoctyes. They found that very small changes in the amount of Activin/Nodal or BMP had profound effects on the proportion of Flk-1+/PdgfR-α+ cells that appeared early on in the differentiation protocol, and that optimization of these concentrations in cultures of human pluripotent stem cells could give rise to structures that contain more than 50% Flk-1+/PdgfR-α+ cells. A major finding by Keller’s team is that different mouse and human pluripotent stem cell lines required unique optimization to produce maximal results, stressing the importance of using differentiation protocols that are in effect customized to individual pluripotent stem cell lines.

β-Catenin Maintains Pluripotency of Stem Cells with Two Divergent Signaling Cascades

Stem Cell and Cancer Research Institute ♦ McMaster University ♦ University of Guelph

Published in Cell Stem Cell, Feb. 4, 2011

It is widely assumed that β-catenin, a key molecule in the Wnt/β-catenin signaling pathway, helps sustain pluripotency through its interaction with TCL/LEF transcription factors. However, recent research shows that β-catenin also promotes pluripotency by complexing with and stabilizing Oct-4, a key member of the transcriptional network that maintains the pluripotent nature of stem cells. Glycogen synthase kinase-3 (GSK-3) has emerged as an important regulatory of pluripotency, in part because β-catenin is one of its primary substrates. After GSK-3 phosphorylates β-catenin it is degraded, which encourages stem cells to exit the pluripotent state and differentiate to other cell types. Dr. Bradley Doble and his colleagues previously showed that mouse embryonic stem cells (mESCs) that are entirely deficient in GSK-3 express very high levels of β-catenin and exhibit a severe impairment in their capacity to differentiate into the three germ layers. In this recent work, Doble and his team hypothesized that hyperactivated β-catenin/TCF was responsible for the pluripotent “lock” that was imposed on mESCs lacking GSK-3 expression. To the surprise of researchers, GSK-3α/β double knock out mESCs still maintained pluripotency even when they stably expressed a dominant negative form of the TCF transcription factor. How were they doing this? Apparently β-catenin can maintain pluripotency independent of functioning TCF. Researchers showed that β-catenin promotes the maintenance of pluripotency by interacting with Oct-4 in a divergent signaling cascade.

Next Generation Gene Therapy for Hemophilia A: Pre-clinical Progress

Queen’s University ♦ Published in Molecular Therapy, Feb. 1, 2011

Researchers pursuing therapies for Hemophilia A have turned to gene therapy for answers but have struggled to provide convincing pre-clinical results. Patients with the disorder have vastly decreased plasma concentrations of FVIII, a clotting factor that prevents blood loss after injury. Although viral vectors can produce the protein following system injection into animal models, its efficacy is compromised by the introduction of neutralizing anti-FVIII antibodies. Researchers hypothesized that the development of neutralizing antibodies was the result of transgene expression in the antigen presenting cells of mice. The solution to this problem was to “hide” the transgene by placing it under the control of a liver-specific promoter. This approach worked in normal mice, however researchers studying mice with hemophilia B still found that an anti-FVIII immune response was mounted in the presence of the new tissue-specific promoter. As a second layer of defense against this response researchers incorporated target sequences into the transgenic construct that had perfect complementarity to hematopoietic-specific miRNA sequences. These target sequences led to suppression of the transgene specifically in hematopoietic cells, including antigen expressing cells, limiting the neutralizing response. Dr. David Lillicrap and his team at Queen’s University have now used a similar approach to produce some very promising results in a mouse model of hemophilia A. A combination of a liver-restricted promoter, a miRNA regulated FVIII transgene, and a pseudotyped viral envelope seemed to do the trick.

Friday Science Review: February 4, 2011

Precious GEMMs: Mouse Models Simulate Metastatic Disease for Tomorrow’s Cancer Therapeutics

Sunnybrook Health Sciences Centre ♦ Published in Nature Reviews Cancer, Feb., 2011

Before cancer therapeutics are moved to the clinic for testing in humans, they must first be assessed in laboratory animals for both safety and efficacy. Developing efficacious therapeutics for cancer treatment is a challenge, by any standard of the word. The process requires animal models of disease that closely simulate similar disease conditions in humans. The introduction of the xenograft mouse model was a large step forward in the development of new treatment regimes for cancer. When carrying out xenograft experiments in mice, researchers transplant human cancer cells into a specific location in the mouse, often subcutaneously, to observe tumour formation and how the tumour responds to treatment with different therapeutics.

Dr. Robert Kerbel and his colleagues at the Sunnybrook Health Sciences Centre reiterate the importance of improving the mouse model for the generation of more effective cancer treatments. With respect to the selection of new drugs for cancer treatment, the predictive abilities of xenograft models suffer from the fact that they fail to adequately simulate the corresponding disease in humans. Human cancer cells, when transplanted subcutaneously into the mouse, grow considerably faster than they do in humans. As a result, xenograft tumours are somewhat hyper-sensitive to chemotherapeutics, which have been designed to target rapidly dividing cells. The location of transplantation plays an important role in how a tumour will respond to treatment. As an example, a tumour found in a human breast will respond differently to cancer treatment than a breast cancer tumour beneath the skin of a mouse. The biological niches in which these tumours reside are quite different, and given the importance the cellular niche plays in cell processes, a difference in niche will translate to a difference in response to therapeutics.

Genetically engineered mouse models (GEMMs) provide a solution to at least some of the problems encountered with the more simple xenograft model. The genetic code of these mice can be altered through the deletion or over-expression of genes that are involved in the tumorigenic processes of specific cancer types. These genetic alterations give rise to tumours in selected tissues such as the breast and lung that are composed of cells and vasculature that are the host’s own. Although GEMMs are a one-up on xenograft models, and have proven valuable for the study of formation and early onset of different cancers, they fall short on one cylinder — primary tumours found in GEMMs rarely metastasize — a hallmark of many cancers. Thus, the clinical predictive power of GEMMs is higher than xenograft models, but is still fundamentally limited.

Dr. Kerbel and his team have addressed this issue using an in vivo selection technique to generate melanoma and breast cancer cells lines that exhibit extensive metastatic capacities following their transplantation into the skin or breast respectively. The selection protocol is rather logical. To create a breast cancer cell line with metastatic abilities a breast cancer cell line is transplanted into the mammary fat pads of an immune compromised mouse. Roughly 4-6 months later some mice will have tumour metastases in the lung. These tumours are dissected, a new cell line is established in vitro, and these cells are then transplanted into the mammary tissue of a second mouse. After two rounds of this selection, mice exhibit extensive metastases to the lung and in some cases, at later stages, the brain. So why is this clinically relevant?

Well, beyond mimicking the normal “metastatic cascade”, treatment of mice that have primary tumours and distant metastases has actually mirrored observations we have made in the clinic for some time. Dr. Kerbel provides a nice example using two monoclonal antibodies. Treating mice with trastuzumab as a monotherapy potently inhibited primary tumours but had very little effect on metastases. Similarly, treatment of mice with a VEGF receptor 2 antibody (DC101) inhibited primary tumour growth but failed to elicit a reduction in the growth of metastases. On the contrary, mice treated with the chemotherapeutic CTX in combination with DC101 inhibited primary tumour growth and the appearance of metastases. This finding is in keeping with the clinical observation that treatment with trastuzumab or DC101 alone as monotherapies has very little clinical benefit for cancer patients, and a combination of antibody and chemotherapy is required for successful treatment in a metastatic setting. We have come a long way with animal disease models and as their predictive powers continue to rise, so should our ability to chose the most effective cancer therapies for the clinic.

TrkC & PTPσ, the Velcro at Neural Junctions

University of British Columbia ♦ Published in Neuron, Jan. 27, 2011

The development of healthy synapses requires a confluence of biological events to occur in harmony at the site where the axon and dendrite meet. The axon of one neuron extends from its cell body towards a dendrite, a short projection radiating from the cell body of an adjacent neuron. Once these two neural components are in close proximity, two key events must occur to drive synaptogenesis. Firstly, “synapse organizing” proteins must help locally recruit pre-synaptic and post-synaptic elements to the ends of the axon and dendrite respectively, and secondly, the axon and dendrite must come into physical contact, a process that is mediated by cell-adhesion molecules. Researchers at the University of British Columbia have discovered a new complex that spans the synapse to bridge axon and dendrite. Dr. Ann Marie Craig and her team used a functional expression screen to identify TrkC, a post-synaptic adhesion molecule, and PTPσ, a high-affinity pre-synaptic receptor of TrkC, which when bound maintain tight synaptic junctions. Neurotrophin receptor tyrosine kinases (Trks) have been known to contribute to nervous system development by interacting with soluble neurotrophins at the post-synaptic membrane. Activation of Trks by neurotrophins leads to signaling cascades that modulate synaptic development. The finding that TrkC interacts with PTPσ on the pre-synaptic membrane is currently the best explanation for why Trks, which are typically catalytic proteins, have cell-adhesion domains and non-catalytic isoforms.

Friday Science Review: January 28, 2011

Cancer’s Byzantine Architecture – The Plot Thickens

Campbell Family Institute for Cancer Research ♦ Ontario Cancer Institute ♦ University of Toronto

Published in Nature, 20 Jan., 2011

In the mid 90s one of Canada’s foremost stem cell researchers, John Dick, made the rather shocking discovery that not all cancer cells are equivalent. Based on his eloquent work we were able to formulate our current thinking on cancer biology, which has it that only a small sub-population of cells within tumours support malignant expansion, while the majority of cancer cells — although dividing — can only divide so many times before they hit cell cycle arrest and begin to senesce. Therefore, to completely eradicate a population of cancer cells, this minority of “cancer stem cells” must be targeted and destroyed. Our historical view on transformation was that single cells accumulate mutations over time, and in a step-wise fashion their genetic contents slowly mutates to the point that the cell itself loses control over its proliferation. Under this model each clone, or descendant of the original transformant, is linearly related. However, recent genomic work in the area indicates that this view is all too simple. It is now apparent that the architecture, the “framework” upon which cancer supports itself, is in fact a complex and branching network of sub-clones that each have the capacity to support tumour growth. Working with human BCR-ABL lymphoblastic leukemia cell lines, John Dick and his colleagues found that many diagnostic patient samples had several genetically distinct leukemia-initiating clones. DNA copy number alteration (CNA) profiling allowed them to reconstruct an evolutionary map of these clones. Transplantation of clones into xenograft models revealed that the predominant diagnostic clone, sometimes, but not always, was associated with the most aggressive growth properties. In some cases, interestingly, minor subclones proved to be the most potent leukemia-initiating cells. Next generation cancer therapeutics are being targeted to cancer stem cells, but now it appears — for these to be effective — they must target not only the dominant cancer stem cell clone, but all of the minor subclones that may be equally, if not more, vicious.

Freeing Systems Biology Data from the Shackles of the 2D Realm

University of Toronto ♦ Published in PLoS ONE, Jan. 10, 2011

One of the great challenges of systems biology will be to integrate multiple data sets, of widely differing scales, into an interactive and visual interface for human interpretation. Visualization is an important element in elucidating the connections between diverse data sets. Only in recent times have platforms existed that have the capacity to weave together large quantities of data into meaningful 3D representations. Historically, visualizations of biological data have been limited to 2D outputs that fail to do justice to the underlying connections between different biological processes. The need for 3D visualization tools is essential. We as humans have lived and evolved in a 3D environment and as a result have adapted a profound capacity to reason and conceptualize along three axes. In addition, biological processes occur within 3D environments, so carrying out analysis of biological data sets in three dimensions is logical. As a solution to this challenge, a group of researchers at the University of Toronto led by Dr. Nicholas Provart have created an open-source template that integrates and visualizes systems biology data as  interactive 3D representations on the world wide web. The group applied their template to the model plant organism Aribidopsis thaliana and have dubbed it ePlant. The platform incorporates proteome-scale protein structure prediction and annotation along with existing -omics scale data, and allows users to evaluate protein structure and function, protein-protein interactions, protein subcellular locations (great visual display here), gene expression patterns, and genetic variation. The result is a program that integrates systems biology data found on the nanoscale with genetic variation found on the kilometer-scale. The open-source nature and flexibility of the ePlant framework circumvents one of the major limitations of current computational systems biology tools — accessibility. ePlant does not require users to download specific data visualization and analysis software to their specific operating system, reducing the learning curve required to grasp the program. Software development on the world wide web also allows for community-driven expansion of systems biology software like that of ePlant, allowing for continued growth and refinement.

Friday Science Review: January 21, 2011

How to Build a Retina — Hope for the Three Blind Mice

Ottawa Hospital Research Institute ♦ Review Published in Stem Cells, Jan. 14, 2011

There have been waves of progress in the stem cell world and regenerative medicine is a field that continues to amaze. In a recently published review, Dr. Valerie Wallace underlines the anatomy and developmental sequence of the retina and provides insight into the biological pathways that can be exploited to re-create retinal cells in the lab. The retina is a light-sensitive tissue found at the back of the eye that contains specialized photoreceptor cells known as cones and rods. After light passes through the lens into the eye it travels through a jelly-like substance in the middle to reach the retina. The retina then converts these photons of energy to a signal that the brain can register as an image. The key to producing functional retinal cells is in exploiting the signaling pathways and chemical cues that lead to their natural formation in the body. In 2009 a paper published in Cell Stem Cell provided evidence that human embryonic stem cell-derived photoreceptors could integrate into the retina and partially repair vision loss in blind mice. This was a pivotal study demonstrating that cell types derived in vitro can potentially be used to rescue vision. There are hopes that similar techniques will be able to treat patients suffering from a variety of retinal diseases, including glaucoma, retinitis pigmentosa, age-related macular degeneration, and diabetic neuropathy. However, as is the case with all prospective transplantation therapies, producing pure, safe, and functional populations of relevant cell types in the lab will be a difficult hurdle to jump — but certainly not one that is impossible to jump. Protocols for the derivation and identification of photoreceptor cells are currently being honed and as we continue to learn more about the developmental pathways that contribute to their formation, these cell populations will move closer to being implemented in the clinic.

Genomic Signature Cracks the Case

Pacific Biological Station ♦ Published in Science, Jan. 14, 2011

In a nice bit of detective work researchers at the Pacific Biological Station in Nanaimo, BC, have figured out the probable cause of the precipitous decline in Canada’s stock of wild salmon. Historically, as many as 8 million sockeye salmon return annually from the Pacific Ocean to spawn in the Fraser River basin. However over the past two decades these numbers have dropped rapidly as salmon die en route to their destination. In 2009 the returns to the river were less than the replacement rate, a finding that spurred a judicial inquiry into the matter. Investigators were able to correlate physiological profiles with failed migration and reproduction by taking non-lethal biopsies of gill tissue from salmon caught in the ocean and tracking salmon with radio transmitters. The gene expression profiles of fish that were successful in making the journey back to the Fraser were then compared with those that perished en route. Researchers found several genes that were associated with survivorship and noted that 60% of fish contained a gene expression signature that was predictive of in-river fate when they were greater than 200km from the mouth of the river. Several genes in the mortality-related signature had known linkages to viral activity, consistent with the finding that fish with this signature also exhibited an up-regulation in inflammatory and apoptotic processes. Researchers attribute the increased mortality of sockeye salmon to viral infection, being exacerbated by the physiological demands placed on salmon as they return from a salt water environment to a fresh water environment and begin their long journey upstream.

The Genome, The Proteome, How About the Tyrosine Phosphatome?

McGill University ♦ Review Published in Nature Reviews Cancer, Jan. 2011

We’re in the era of “-omics” and as we continue to explore the microscopic world within us we find more and more families to apply the suffix to. Protein tyrosine phosphatases (PTPs) play an important role in the regulation of numerable biological processes that are intertwined in the development of cancer. Dr. Sofi Julien and her colleagues at the Goodman Cancer Research Centre and Department of Biochemistry at McGill University have prepared an impressive review on the “human cancer tyrosine phosphatome”. The work focuses on the genetic and epigenetic alterations that may lead to loss or gain in function of PTPs that are involved in cancer formation, and provides figures illustrating such things as the location of PTP genes on chromosomes, the location of mutations in PTPs, and proposed mechanisms of both oncogenic and tumour suppressor functions. Interestingly, PTPs have modes of action that can both cause and prevent cancers depending on the cellular context. PTPs exert their effects by removing phosphate groups from target proteins, and depending on the type of protein that becomes dephosphorylated, the resulting signaling cascade can promote or suppress tumour formation. PTPs counter the effects of protein tyrosine kinases (PTKs) which add phosphate groups to target proteins as opposed to removing them. The activity of PTPs and PTKs exist in a sort of equilibrium within the body and shifts in this balance can have detrimental effects. Generally speaking members of the kinase family are considered to be oncogenic because overzealous phosphorylation activity, particularly activation of growth receptors on the extremity of cells, can lead to rapid and uncontrolled cell proliferation. Since the late 1980s, when the first true phosphatase was discovered, more than 100 have been identified by scientists. PTPs have the potential to be used as prognostic indicators for different cancer types. Investigators are also hoping that PTPs may prove to be effective drug targets for cancers where PTP hyperactivity is known to contribute to formation and onset of disease. PTP inhibitors, including natural compounds, small molecules, silencing RNAs, and anti-sense molecules are all under development. The only advanced clinical trial of an anti-PTP therapeutic is ISIS’s 113715 which targets PTPN1. The antisense molecule successfully made its way through a phase II clinical trial for diabetes. PTPN1 has been identified as an oncoprotein in a mouse model of breast cancer suggesting that it may also have utility in an oncology setting.

Friday Science Review: January 14, 2011

The Eukaryotic Tree of Life Expands

Dalhousie University ♦ Published in PNAS, Jan. 4, 2011

Photosynthetic marine organisms carry out roughly half of the primary production on the planet today. Tracing the lineages of these tiny creatures has helped us document eukaryotic evolution and draw conclusions on the events that led to their current distribution and the distribution of the genetic content hidden within them. A new lineage of photosynthetic algae, being referred to as rappemonads, has been discovered by Dr. John Archibald’s lab group in the Department of Biochemistry and Molecular Biology at Dalhousie University. Phylogenetic analysis using operons from plastid ribosomal DNA indicates that this new group is indeed evolutionarily distinct. Furthermore, scientists revealed that the habitat distribution of rappemonads is wide; environmental DNA sequencing in the North Atlantic, North Pacific, and at European fresh water sites suggests an extensive diversity. Although flare-ups of this new species are rare, they are believed to be able to form transient blooms. Quantitative PCR analysis was able to detect large quantities of rappemonads rRNA in the Sargasso Sea. Discoveries of this nature bring to mind the Sorcerer II expedition — launched by Craig Venter in 2004 — where researchers traveled the world’s oceans to discover new microbial species. The field of environmental genomics is in its infancy, and has the potential to help us alleviate some of our environmental issues and elucidate many aspects of biodiversity and evolution.

Novel Vaccine Delivery Formulation Protects Against Respiratory Pathogen Challenge

Institute for Biological Sciences, NRCC ♦ Published in PLoS ONE, Dec. 29, 2011

Mucosal surfaces represent an excellent opportunity for microbial pathogens to invade the body and give rise to infections. Currently many systemic vaccines targeting these pathogens fail to elicit adequate mucosal immunity in the host. It is for these reasons that Dr. Wangxue Chen and his colleagues at the NRCC are developing mucosal vaccines that specifically target these entry points. Creating long lasting and memory boostable immune responses has proven difficult with mucosal vaccines however, and they typically require an adjuvant, or delivery vehicle, to be successful.  The team at NRCC has found that intranasal immunization of mice with a cell free extract of Fransicella tularensis has much more pronounced effects when it is paired with archael lipid mucosal vaccine adjuvant and delivery (AMVAD). The technology incorporates cell free extract, from the organism against which immunity is desired, into liposomes which can then be delivered as a vaccine. Mice receiving the AMVAD/extract preparation had lower pathogen burden in the lungs and spleen, longer mean time to death, and significantly greater overall survival than mice that received just the cell free extract or naive mice receiving no vaccination.

Oxidative Stress of Surrogate Tissues Mirrors that of the Prostate

McMaster University ♦ University of Toronto ♦ Published in PLoS ONE, Dec. 28, 2011

Researchers believe that surrogate androgen regulated tissues from the same host can be used to determine the oxidative stress (OS) status of the prostate. Androgens have long been known to drive the formation of prostate cancer. Oxidative stress is regulated by androgens, so reducing OS is a key target in the prevention of prostate cancer. Using a mouse model researchers show that the level of prostatic OS is correlated with the OS of Dermal Papillary Cells, a cell type found in hair follicles, and also the salivary glands – two exocrine glands that express the androgen receptor and are morphologically similar to the prostate. Determining the OS status of the prostate and patient response to prevention strategies directly, would require taking a biopsy sample from the prostate itself. Thus, the findings of Dr. Jehonathan Pinthus and his team at McMaster University could have great implications for the non-invasive and indirect evaluation of prostate OS status and patient response to prevention strategies.

Friday Science Review: January 7, 2011

Symmetry Saves the Day

University of Toronto ♦ Published in Stem Cells, Dec. 29, 2010

One of the hallmarks of stem cells is their ability to maintain the stem cell pool indefinitely through the process of asymmetric division. When they divide they give rise to one slightly more differentiated cell and one daughter stem cell identical to the original. By carrying out cell division in this manner, stem cell populations, at least in theory, are capable of living indefinitely. David Piccin and Cindi Morshead — researchers at the Donnelly Centre for Cellular and Biomolecular Research — discovered that the Wnt (pronounced ‘wint’) signaling pathway is involved in damage response in neural stem cells found in the brain. Using a mouse model Piccin and Morshead show that when neural stem cells sense it is time to replenish the stem cell population, for example during the period following a stroke, Wnt signaling contributes to a signaling cascade that promotes symmetric division. When stem cells divide symmetrically they produce two identical daughter stem cells rather than one daughter stem cell and a differentiated progenitor, ensuring that the stem cell pool does not become depleted.

H5N1 Vaccine Derived from Tobacco Plants Shows Results in the Clinic

Medicago Inc. ♦ Published in PLoS ONE, Dec. 22, 2010

Egg-based vaccine manufacturing failed to live up to its promise of rapidly producing large quantities of live vaccine for control of viral outbreaks. During the recent H1N1 influenza pandemic only 3 million doses of live vaccine had been produced by the 5 month mark, when 60 million had been expected. Canadian biotechnology company Medicago Inc. has come up with an all together different approach that could make fast and efficient vaccine production a reality — a plant-based manufacturing technology that produces influenza vaccines using Nicotiana benthamiana. At the core of the vaccine technology is something known as a “Virus-Like Particle” (VLP). VLPs are small entities containing the hemagglutinin protein of H5N1, and are produced by infecting tobacco plants with an Agrobacterium inoculum containing an H5 expression cassette. VLPs are then harvested from the aerial portions of the plant. Although a VLP resembles a viral particle, it lacks the genetic content within, thus is replication defective and non-infectious. Another aspect differentiating Medicago’s approach is that the technology only requires the genetic sequence of the virus, not an actual sample, as is the case with technologies using inactivated virus in vaccines. In a preclinical study led by Medicago’s Dr. Louis Vezina, researchers show that a VLP vaccine could induce cross-reactive antibodies in ferrets. After challenging the animals with lethal doses of H5N1 researchers observed reduced pathology and suppressed viral loads in vaccinated animals. The paper also reports on clinical results: a phase 1 trial of the H5 VLP vaccine in healthy adults between the ages of 18 and 60 revealed that the plant-derived vaccine was tolerated well at all doses and had strong immunogenicity as detected by microneutralization assays. These results taken together hold promise for Medicago’s plant-based manufacturing technology. Another plus? The vaccine can be produced in 3 weeks of sequence release! This is no doubt why DARPA made a non-repayable contribution of $21 million to Medicago back in August to build a 90,000 square foot cGMP facility in North Carolina for VLP vaccine production.

Friday Science Review: December 31, 2010

Just a couple papers to squeeze in this year before the clock strikes 12. I look forward to 2011 and the research it will bring in the Canadian realm. For those readers heading out tonight for some fun on the city, enjoy! More science reviews to come in the new year..

Porcine Adenovirus PAV3, A Novel and Promising Candidate for H5N1 Protection

National Microbiology Laboratory, Winnipeg ♦ Published in PLoS ONE, Dec. 16, 2010

Researchers have provided evidence that suggests a porcine adenovirus, PAV3, has greater vaccine efficacy than the human adenovirus AdHu5 in protecting against H5N1. An avian influenza H5N1 mouse model was used to compare immune response and protection following vaccination with the two different vectors. Mice that were vaccinated with a replication defective PAV3 vector carrying an H5N1 antigen expressed higher concentrations of neutralizing antibody post-vaccination and had stronger cellular immune responses than mice vaccinated with AdHu5. After challenging vaccinated mice with H5N1 infection, Dr. Gary Kobinger and his team demonstrated that mice inoculated with PAV3 showed higher overall survival. Another notable finding was that the porcine adenovirus did not become significantly neutralized when exposed to a pool of antibodies generated from 10,000 humans.

Study of Human Heart Microsomes Gives Insight into Cardiac CYP450s

University of Montreal ♦ Published in PLoS ONE, Dec. 14, 2010

Enzymes from the cytochrome P450 (CYP450) superfamily play an important role in drug metabolism. Variation in CYP450 isoforms can lead to inter-subject and inter-organ variability in drug metabolism, thus their study is crucial to understanding the metabolism of specific drugs. Dr. Jacques Turgeon and his colleagues at the University of Montreal gathered data on CYP450 mRNA levels in left and right ventricular samples taken from the explanted hearts of patients with end-stage heart failure. Samples were processed in the lab to extract microsomes, small vesicle-like structures composed of endoplasmic reticulum that contain large quantities of CYP450s. Among the interesting findings of this body of work is that CYP2J2 was the most abundant isoform found in cardiac tissue samples. Levels of CYP450 mRNA were similar across ischemic and non-ischemic samples and between left and right ventricles. Another principal and interesting finding was that the stereoselectivity of cardiac CYP450s was reversed compared to those in the liver. After exposing heart microsomes to the calcium channel blocker verapamil, higher levels of CYP450-dependent metabolites were observed in the presence of the R-enantiomer.

Friday Science Review: December 24, 2010

Given that the UN Climate Change Conference has just wrapped up in Mexico, I thought for the Christmas edition of the FSR I would lay out some articles from Nature focused on global warming and its impact on one of Canada’s most iconic animals — the polar bear. University of Alberta’s Andrew Derocher reports on recent findings that suggest we can curb the polar bear’s extinction, but only if policy-makers move quickly. Steven Amstrup of the Alaska Science Center created a number of greenhouse gas emission scenarios and examined them within a projection model of sea-ice loss. The results indicated that mitigation has the potential to greatly improve the polar bear’s situation in the snowy north. Amstrup believes that reducing emissions by sufficient amounts could increase the abundance of polar bears and broaden their distribution.

Melting ice also places the genetic diversity of Arctic animals at risk. A recent Nature commentary piece describes how the loss of habitat forces Arctic animals into environmental niches they would otherwise remain clear of, which can lead to interbreeding with different species. Brendan Kelly of the National Marine Mammal Laboratory in Alaska refers to this effect as a “melting pot”. In 2006, a white bear with brown patches was shot and killed by hunters in the Arctic. Genetic analysis would later show what many feared, that the animal was a hybrid between a polar bear and a grizzly (the polar bear in the photo above hasn’t been rolling in the mud, it’s a suspected hybrid). This year an even more unlikely event occurred when hunters in the Canadian north shot and killed a 2nd generation hybrid bear — its mother was a hybrid and its father a grizzly.

The outcome of the recent UN climate talks was positive, with unanimous adoption of the Cancún agreement. Developing countries will also be required to take heed to global warming policy and act to reduce greenhouse gas emissions. Steps are being made in the right direction but execution and adherence will be essential to seeing results that benefit the earth and its animal populations in the coming decades.

As we approach year end I thought I would also reference you Nature’s “2010 Gallery: Images of the year“, which provides a fascinating look at some of the natural wonders that amazed (and scared) us over last 12 months. I should also point you towards an excellent article on nanomedicine recently published in NEJM; written by Dr. Betty Kim of the Institute for Biomaterials and Biomedical Research and the Terrence Donnelly Centre for Cellular and Biomolecular Research in Toronto, the review covers the properties of nanomaterials and the myriad in vivo and  in vitro applications of these tiny tools.

Friday Science Review: December 17, 2010

I’ll begin the FSR this week with a few comments regarding some investigational work coming from the McLaughlin-Rotman Centre for Global Health.   Professors Dr. Peter Singer and Dr. Abdallah Daar, and PhD student Ken Simiyu, traveled to Africa to better understand why commercialization in the biotechnology and healthcare industry has been so poor of late.

Stagnant Technologies Need Stimulus

University of Toronto ♦ Published in Science, Dec. 10, 2010

After visiting some 23 academic institutions in six countries and interviewing 39 scientists, researchers have dug up some of the underlying issues preventing Africa’s biotechnological innovations from migrating to commercial success. Although previous studies in Africa have analyzed health innovation at the country level there has never been a systematic evaluation highlighting the troubles of specific technologies. Some of the technologies identified in the study include traditional plant products, new chemical entities, diagnostics, vaccines, and medical devices. In their travels the group came across some very interesting technologies indeed; researchers at the University of Ghana are developing a visually readable point of care diagnostic that uses monoclonal antibodies to detect the malaria parasite in urine; other work from Tanzania’s National Institute for Medical Research is being invested in the development of novel extraction techniques, specifically those to extract and purify artemisinin from the plant Artemisia annua for the preparation of derivatives to fight artemisinin resistance in malarial therapy. So why aren’t these technologies making a move towards market? A number of reasons. The mindsets of many researchers interviewed were simply not commercially oriented, with most scientists focusing on teaching and publishing to disseminate knowledge. Finding funding for validation studies of early stage technologies is another issue. African scientists need support from institutional investors but there are very few African funds in existence that support the biotechnology and healthcare space. Other issues identified by interviewees included a lack of commercially oriented government policy, poorly understood intellectual property regimes, and regulatory red tape. Peter Singer previously identified three areas that would help spur technology development in Africa: proof-of-concept funds, networks to link scientists and entrepreneurs together, and innovation centres that provide shared research infrastructure. Some have proposed establishing ‘Life Sciences Innovation Centres’ throughout Africa. These would serve a similar purpose as MaRS, here in Toronto, and the newly proposed Clerk-Maxwell Centres in the UK, with the goal of uniting researchers, industry, and entrepreneurs to accelerate commercial development of life science assets. This integrative approach is catching on, and could be the ingredient that will remedy the static nature of Africa’s commercial environment in the life sciences sector.

RD3 at the Root of Congenital Blindness

University of British Columbia ♦ Published in PNAS, Dec. 7, 2010

The protein RD3, previously of unknown function, has been implicated in the development of Leber Congenital Amaurosis Type 12 (LCA12). The disease is characterized by rapid degeneration of the photoreceptor cells during fetal development leading to blindness at birth or in the first year of life. Dr. Robert Molday and his team at the Centre for Macular Research show that RD3 interacts with two different forms of guanylate cyclase, GC1 and GC2, mediating their export from the endoplasmic reticulum. GC1 and GC2 are essential for the production of cGMP — a secondary messenger of phototransduction — and in their absence cGMP production is impaired. Dr. Molday believes that LCA12 may be caused by cGMP deficiency which leads to constitutive closure of cGMP gated calcium channels. Proper gradients of calcium across the membranes of photoreceptor cells is likely required for their long-term survival.

New Target for Chronic Pain Unveiled

University of Toronto ♦ Published in Science, Dec. 3, 2010

Synaptic plasticity is the ability of neural connections to vary in strength based on the extent of use or disuse of a neural pathway. This characteristic of the nervous system is key to the process of learning and memorizing sensory experiences, and it is also believed to play a role in pathological pain. Most researchers have focused on proteins that lead to synaptic plasticity as opposed to those that maintain synaptic plasticity over the long-term. A new study led by Dr. Min Zhuo implicates protein kinase M zeta (PKMξ) in the maintenance of chronic pain. Peripheral damage in a mouse model upregulated PKMξ in the anterior cingulate cortex, a region of the brain known to be involved in the onset of chronic pain. Zhou went on to show that microinjections of a PKMξ inhibitory peptide ZIP into the anterior cingulate cortex dampened synaptic potentiation and behavioural sensitization. This research uncovers an excellent target for chronic pain.

Trends Update — Social Media Reshaping Healthcare: Twitter as a Public Health Surveillance Tool for the 21st Century

We have been following innovative uses for social media in the biotech and healthcare industry here on the blog. Recently, a comprehensive paper was published in PLoS ONE outlining the use of “infoveillance” tools on the web to track the public response to the H1N1 epidemic. Dr. Gunther Eysenbach and Cynthia Chew, both researchers at Toronto’s Centre for Global eHealth Innovation, mined and archived over 2 million Twitter posts between May 1 and December 31, 2009. After carrying out an in depth analysis of these “tweets”, they validated Twitter as an effective medium to capture real-time content, sentiment, and public attention trends. Infoveillance methods include data mining, aggregation, and categorizations of online text and together form the toolkit for the new study of “infodemiology”. In the paper, Eysenbach points out that Twitter is particularly amenable to textual mining and analysis due to the concise nature of tweets that users share with their respective followers.

The concept of infodemiology began to crop up in the early 2000s when several different researchers began playing with the idea of using Internet health-related searches to provide epidemiological data that could be used to inform public health. In 2004, Eysenbach became interested in tracking flu-related searches using online syndromic surveillance. Historically, syndromic surveillance systems have typically relied on data from patient encounters with health professionals. But what if we could track the health concerns of citizens before they ever see a physician? It turns out we can. The study of infodemiology on the World Wide Web has the potential to provide automatic, continuous, and virtually real-time snapshots of public opinion and behavioural trends. In the context of public health this means capturing public health concerns at their earliest stages and even predicting major influenza pandemics weeks before they happen.

In 2006 Eysenbach published findings from a rather clever experiment he had completed over the 2004/2005 flu season. In order to track the number of people in Canada that were searching for either “flu” or “flu symptoms” he created an ad “campaign” through the keyword-triggered advertising program Google AdSense. For the purposes of optics the flu keywords led searchers to an advertisement that linked them to a generic patient education website after a click. Eysenbach then gathered FluWatch data, including influenza cases, positive lab test results, and influenza-like illness reported by sentinel physicians (“ILI-SPR”) around the country, and correlated these disease surveillance metrics with his Google advertisement data. Incredibly, Eysenbach found that clicks on the flu advertisement he had created correlated more strongly and in a more timely fashion (statistically significant on both accounts) with influenza cases and positive lab test results than did the ILI-SPR data. In a nutshell, his online experiment was more accurate at predicting rises in influenza cases than was the nation-wide sentinel physician program.

Three years later in 2009 a research paper funded by Google was published in Nature describing an influenza surveillance system piggy-backing on the popularity of certain Google search queries. The model underlying the surveillance system was generated by processing hundreds of billions of previous individual Google searches stored in web search logs. (Despite Eysenbach’s earlier work being cited in the Google paper, most journalists failed to recognize that Google’s idea wasn’t entirely new). Today Google Flu Trends can be accessed online to follow worldwide estimates of influenza-like activity. Google may have to go back to the drawing boards and refine its model however, as a new study published this past summer shrouds the accuracy of the system in some doubt. Researchers at the University of Washington evaluated Google Flu Trends against the gold standard positive influenza virus infection and its accuracy came up short – about 25% short.

The new H1N1 Twitter study reaffirms Eysenbach’s status as a visionary in the field of infodemiology. With “Web 2.0” upon us, and tsunamis of user-generated content flooding the web, the Internet “has made measurable what was previously immeasurable” in Eysenbach’s words. What we could not measure 10 years ago due to the (comparatively) static nature of the Internet, is now readily measurable with infoveillance tools. In the context of H1N1 Eysenbach says:

“H1N1 marks the first instance in which a global pandemic has occurred in the age of Web 2.0 and presents a unique opportunity to investigate the potential role of these technologies in public health emergencies.”

To carry out analysis of tweet content in the H1N1 study Chew and Eysenbach used an open-source infoveillance system known as Infovigil (Eysenbach’s own creation) that automatically and continuously dissects textual information from Twitter. They created a “codebook” with three primary variables: 1) tweet content, 2) mode of expression, and 3) type of link posted, if any. Each of these categories had several subcategories allowing for good separation of different tweet “types”. The study had some interesting findings. Over the duration of the study the relative proportion of tweets using “H1N1” increased from 8.8% to 40.5%, indicating that the public gradually began to adopt the WHO-recommended terminology as opposed to “swine flu”. With respect to tweet content, personal accounts of H1N1 increased over time while humorous content declined, indicating that the public’s perception of the subject became more serious. The public attention was aroused in certain instances, especially following the WHO pandemic level 6 announcement on June 11, 2009, which gave rise to a large spike in tweets. Only 4.5% of tweets were identified as misinformation.

Overall the study is a nice proof of concept and displays the fact that Twitter is a rich source of public opinion for the health authorities. Infoveillance can be used in the future not only for capturing sentiment, experiences, and behavioural trends, but importantly for tracking misinformation and identifying the informational needs of the human population. More studies of this kind should elucidate the value that social media will have for knowledge translation research and help refine the precision and accuracy of infoveillance tools for future infodemiology studies.

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