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Category Archives: Mark Curtis

Friday Science Review: February 10, 2012

Automated Regulation of Inhibitory Feedback Signalling Leads to Rapid and Robust Expansion of Cord Blood-Derived Hematopoietic Stem Cells

University of Toronto ♦ Genomics Institute of the Novartis Research Foundation ♦ Heart and Stroke/Richard Lewar Centre of Excellence ♦ McEwen Centre for Regenerative Medicine

Published in Cell Stem Cell, February 3, 2012

The greatest current justification for the storage of cord blood stem cells, in the setting of both the private and public cord blood bank, is for hematopoietic stem cell transplantation (HSCT) for reconstitution of the bone marrow compartment; most often following intensive chemotherapy regimens that ablate the bone marrow completely.

Cord blood is a viable source of hematopoietic stem cells (HSCs), however a single cord blood unit in its original form only contains a small quantity of these cells. It has been found that the most critical factor in patient survival following HSCT is administering a threshold cell dose (roughly 30 million cells per kilogram patient) that must be met or surpassed in order to achieve successful engraftment and reconstitution of the bone marrow. As a result, the majority of clinical studies utilizing cord blood stem cells for HSCT have been reserved for the paediatric population. However, even in children, successful engraftment and recovery is by no means a given. Despite the widespread storage of cord blood units in public banks that can be HLA-matched to recipients, and the numerable benefits of this source of HSCs over others, cord blood has yet to become a viable solution for HSCT.

One treatment paradigm under current investigation in the clinic is double cord blood transplant, wherein two units are transplanted simultaneously in order to boost cell number. The shortcoming of this approach is the difficulty in acquiring two units that are HLA-matched to the recipient. Although cord blood is better tolerated by the host’s immune system following transplantation, relatively speaking, and typically exhibits lower levels of graft versus host disease than other sources of HSCs, finding two adequately safe units for a single patient is no trivial task. With a cord blood unit ringing in at $30,000 a hit (from a public bank) the economics of this approach are also prohibitive.

A second approach has been to expand cord blood units in the lab prior to transplantation. This process increases the number of HSCs in the unit by several-fold; the results depending on the protocol and hands at work. Preclinical studies show that expanded cord blood stem cells can reconstitute the bone marrow compartment in immune compromised mice. The medical community has begun preliminary studies in the clinic mixing expanded cord blood stem cells with unexpanded (mixed because there is currently not enough definitive evidence to suggest that expanded cord blood stem cells retain repopulating activity). The results of these studies so far have failed to show that the expanded product contributes significantly to engraftment and recovery. Expansion protocols have become iteratively better, however limitations on HSC number, and their ability to accurately home to the bone marrow compartment and engraft, have prevented expanded units from reaching their potential in the clinic.

Expansion protocols modulate molecular mechanisms that regulate stem cell fate and proliferation in order to maximize the number of HSCs produced. Two approaches have primarily been used. The first, cytokine-driven expansion, utilizes molecular messengers relevant to the bone marrow niche. These proteins interact with surface markers on HSCs, triggering pathways that help reinforce cell fate decision towards the HSC identity. Cytokines are amenable to expansion as cells can be grown in 3-dimensional space, often in bags, which allows for easier scale-up. This being said, it is arguable that the approach is flawed, as it fails to truly recapitulate signaling mechanisms in the bone marrow niche where cell-to-cell contact is critical for the maintenance of different HSC pools.

The second approach, stromal-driven expansion, expands HSCs in the presence of a second population of cells known as stromal cells. While this cell culture approach provides a microenvironment that more accurately reflects the niche an HSC would experience inside the body, it is exceedingly difficult to scale-up to produce clinically relevant cell numbers.

Both approaches struggle in producing large numbers of clinically relevant populations of HSCs. Emerging data supports the idea that not all HSCs in the bone marrow compartment are equivalent. Slight differences in their gene and protein expression stratify them into classes that behave differently in terms of their capability to home and engraft. Some HSCs exhibit the classical HSC markers but only retain repopulating capacity transiently. Other rarer HSC populations exhibit a unique and specialized capacity to form colonies over the long-term. These stem cells, known as long-term repopulating HSCs (LTR-HSCs), are the cells that expansion protocols must produce if they are to create a cord blood product that is useful to humans in the clinic. In addition, neither approach accounts for the production of HSC progeny that amass within the cell culture system. These differentiated cells produce high concentrations of inhibitory feedback molecules that prevent HSC proliferation.

A transformative development in this space is the advent of a cell culture platform that enables rapid expansion of hematopoietic stem cells from a cord blood unit at some of the highest levels ever achieved. Developed by Peter Zandstra at the University of Toronto, this closed-system approach utilizes a controlled fed-batch media dilution strategy to reduce concentrations of proteins that inhibit stem cell proliferation. Within 12 days, LTR-HSC populations can be scaled-up to 11 times their original number while retaining their capacity to self-renew and differentiate into cells of multiple lineages. At its core, the platform hinges on the concept that HSC self-renewal and differentiation are regulated tightly by secreted factors that either promote or inhibit stem cell proliferation.

Zandstra’s group took a computational approach based on the effects of feedback signaling to design the expansion protocol. Measurements of secreted factors were taken from previously established in vitro growth conditions to identify factors that had inhibitory effects on HSCs. Computational simulations were then performed to model the effects that inhibitory proteins would have on stem cell population dynamics. Simulations depicted an accumulation of predominantly inhibitory proteins within the system. Hence, the group rationalized that media exchange would be a key advance in stimulating stem cell expansion. Further investigation with the simulation predicted that a fed-batch process (continuous input of new media) would outperform both perfusion (continuous input of new media and output of old media) and frequent full or partial media exchange. The hypothesis generated was then tested utilizing an automated media delivery system, which confirmed that the fed-batch approach led to a significant increase in the absolute numbers of various HSC subpopulations over other media exchange processes.

A critical component of this study was evaluating the increase in expansion of LTR-HSCs. The only means in which to do this is to carry out transplantation studies in immune compromised mice. Repopulation of the bone marrow compartment was quantified by judging the extent of the contribution of human cells to hematopoietic reconstitution. All of the mice that experienced successful repopulation of the bone marrow exhibited multilineage differentiation, as indicated by the presence of human cells from the myeloid, lymphoid, and erythroid lineages, and the presence of T-cells. Importantly, human cells from repopulated mice could be transplanted to reconstitute the bone marrow of secondary mouse recipients, confirming the long-term engraftment potential of the HSCs at hand.

Limiting dilution analysis was used to quantify the expansion of LTR-HSCs. In fresh cord blood the frequency of LTR-HSCs was roughly 1 in 14,700. After 8 days of growth in the fed-batch system this expansion was increased by 7.6-fold to a frequency of 1 in 1,940. And finally, after 12 days the expansion had increased by 11-fold to a frequency of 1 in 1,334.

Zandstra has created a cell culture technology that rapidly and cost-effectively expands clinically relevant populations of blood stem cells that retain the ability to engraft and contribute to hematopoietic reconstitution over the long-term. A technology of this nature is truly enabling. Not only does it provide new potential to the hundreds of thousands of cord blood units currently stored in public stem cell banks, it ensures, at least in the eyes of HSCT with cord blood stem cells, that cord blood units stored in the future will be put to good medical use. On a high level, the technology is also a platform approach to the problem of scaling up any number of different stem cell types for cell therapies in the future.

An interesting innovation for the future of this technology would be the addition of a device that can measure the concentration of inhibitory proteins within the system as cell growth occurs. If this could be achieved in real-time, it is conceivable that media input could be regulated to create a dynamic stem cell expansion environment. This would be highly fitting for the expansion of stem cells from cord blood units, as the protocol would be tailored to every expanded unit; all of which are different in cellular composition and genetic make-up.

Friday Science Review: February 3, 2012

Pathogenesis of Paediatric Glioblastoma Multiforme

McGill University ♦ Genome Quebec Innovation Centre

Published in Nature, January 29, 2012

Researchers have not only discovered the first recurring mutation in a human histone but have uncovered a key pathway involved in the formation of paediatric glioblastoma multiforme (GBM). This highly aggressive form of cancer is almost always lethal. Previously acquired gene expression patterns suggest that the mechanisms underlying GBM formation in children and adults are different. Mutations involved in the pathogenesis of GBM were identified by sequencing the exomes of 48 paediatric GBM samples. A chromatin remodelling pathway involving the histone H3.3 and the genes ATRX and DAXX seems to be at the heart of transformation. Researchers found that mutations in H3F3A, the gene encoding H3.3, lead to amino acid substitutions in the histone tail, a portion responsible for key regulatory post-translational modifications. In addition, mutations in ATRX and DAXX, both part of a chromatin remodelling complex that incorporates H3.3 histones at telomeres, were also identified in many of the patient samples. Subsequent screening of a large cohort (n=748) of gliomas showed that H3F3A mutation is frequently found in GBM and is highly specific to children.

The mutations identified in this study were associated with elongated telomeres. During the normal aging process telomeres shorten over time, and in a sense are a ‘biological clock’ that dictates the length of a cell’s life. Eventually, after many cell divisions, telomeres reach a critically short length and the cell undergoes senescence and/or programmed cell death. This is one mechanism by which the human body has evolved to prevent cells from accruing enough genomic mutations to undergo malignant transformation. Telomere elongation allows cells to live beyond their normal biological lifespan. Cells with elongated telomeres become dangerous as they are allowed to continue to live in the presence of mutational ‘build up’. Upregulation of the enzyme telomerase, which helps maintain telomere length through the addition of DNA to the ends of chromosomes, is also associated with cellular immortalization.

Friday Science Review: January 27, 2012

An Evolving Concept of Oncolytic Viruses

University of Ottawa ♦ Ottawa Hospital Research Institute ♦ Ontario Cancer Institute ♦ University of Toronto ♦ University of Otago

Published in Molecular Therapy (npg), January 24, 2012

Oncolytic viruses were originally engineered to impose direct damage to tumour cells through infection, replication, and subsequent destruction of cancer cells via cell rupture. Many of today’s oncolytic viruses aim to do this, however it has become apparent that ‘next generation’ oncolytic viruses, those with the greatest efficacy, will be the viruses that not only destroy cancer cells physically but stimulate a strong innate immune response against cancer cells to continue the onslaught following initial infection.

This concept of inducing immune response is not novel. By their nature, oncolytic viruses offer the opportunity to deliver therapeutic genes at the time of infection. Indeed, previous studies have shown that viruses can be engineered to deliver cytokine genes into tumour cells. These cancerous cells then act as factories creating a protein product that leads to their own immune destruction. Suicide.

But researchers have now discovered that it may not be necessary to engineer viruses to do this. Certain viruses are capable of generating such an immune response simply through the presence of the viral particle itself. One example of such a virus is Parapoxvirus ovis, or ORFV. Researchers at the University of Ottawa found that injecting the virus into mice led to a significant upregulation of T cell response, including both CD4+ and CD8+, and an accumulation of B cells, natural killer cells, and various cytokines with anti-tumoural activity.

But there’s more to the story. ORFV infection does not lead to disease in animals, making it an excellent candidate for an oncolytic therapeutic. The real beauty though — the differentiative aspect of this virus — is that ORFV, even in the presence of antibody against the virus, continues to reinfect cancer cells. Animals seem to have a very short-lived immunity against the virus, allowing for an attenuated therapeutic activity unlike any found so far in this field.

Friday Science Review: January 20, 2012

Genetic Basis of Jr(a)- Phenotype Discovered

University of Manitoba ♦ Published in Nature Genetics, January 15, 2012

The medical community has been aware of the Jr(a) antigen on red blood cells for quite some time. Roughly 40 years ago it was shown that a small group of individuals created antibodies against this protein motif. In the presence of normal red blood cells the antibodies produced by Jr(a)- individuals react quite vigorously. During transfusion it is important that donor blood does not contain Jr(a) antibodies because they can lead to negative transfusion reactions that are harmful to the recipient. In this recent study, investigators isolated the genetic component responsible for the Jr(a)- phenotype. Carrying out single nucleotide polymorphism analysis, researchers discovered a nearly 400 thousand base pair null region in Jr(a)- individuals that contained the gene ABCG2.

Cutting to the Core of Systemic Immune Response

Ontario Cancer Institute ♦ Published in Science, January 13, 2012

The human body has become adept at regulating pathogens. However, this evolutionary trait also has its down sides. Extreme innate immune response can cause systemic inflammatory reactions that can prove fatal. This recent study out of Tak Mak’s lab identifies a mechanism by which animals induce innate immune response following exposure to foreign pathogens. Tumour necrosis factor alpha (TNF-α) mediates septic shock through its release from cell membranes. This release process is regulated by an enzyme known as TNF-α convertase. Researchers discovered that this enzyme’s maturation and trafficking is controlled by iRhom2. Sure enough, mice deficient in iRhom2 displayed an ability to circumvent lethal doses of bacterial lipopolysaccharide.

Conditions characterized by systemic immune response are some of the hardest to go after in the clinic. As an indication, sepsis has been a graveyard, with only a few of some 40 clinical studies showing any efficacy in the last two decades. Identifying general systemic pathways, like that involving iRhom2, will be critical in creating therapeutics for these complex conditions in the future.

Friday Science Review: January 13, 2012

Disruption of Gatekeeper Genes Causes Two-fold Mutation

University of Toronto ♦ Published in EMBO, January 10, 2012

Certain genes have a critical role in maintaining the stability of the genome by exerting a certain control over DNA metabolism. Researchers at the Donnelly Centre in Toronto have discovered that disruption of these so called ‘gatekeeper’ genes has consequences beyond disregulating the direct influence they have on genomic stability. As expected, perturbing gatekeeper genes led to spontaneous DNA damage, however, this damage became amplified downstream. Measurements of ribonucleotide reductase (RNR) in budding yeast showed that expression of this enzyme increased as DNA damage occurred, and that RNR activity was associated with an increase in dNTP pools. A second wave of mutation occurred as yeast cells were then capable of synthesizing DNA in the presence of hydroxyurea in the next S phase.

Movement and Breathing, Where’s the Link?

University of Montreal ♦ Published in PNAS, January 10, 2012

As we exercise the rate at which we respire increases, and can do so dramatically. However, there is much to be elucidated in terms of the neural connections that elicit the body’s breathing response to movement. To interrogate this matter, researchers used the lamprey as a model. They first identified the region of the brain that was associated with an increase in respiration. In lampreys, stimulation of the mesencephalic locomotor region (LMR) increased the rate of respiration. Researchers then used a technique called ‘patch-clamp’, that is capable of taking electrophysiological recordings from single neurons, and used it to show that neurons in the dorsal region of the MLR are directly connected to a respiratory generator. To cross-check their findings, an inhibitor of the innervated region of the respiratory generator was used to show that inhibition at this site did indeed reduce respiration.

Friday Science Review: January 6, 2012

MSC Anti-Immune Power

McGill University ♦ Published in Molecular Therapy (npg), January 2012

Mesenchymal stem cells have great potential as a source of therapeutic cell types for transplantation because they are capable of differentiating into bone, fat, cartilage, and muscle. Some reports show they even have the ability to differentiate into neural cells. The research community is beginning to learn, however, that perhaps the most interesting application of MSCs will be as a suppressant for the immune system. MSCs exhibit the unique ability to moderate T cell response. As a result, they are being investigated in clinical studies for treatment of disorders characterized by inflammation and autoimmunity. Rheumatoid arthritis is a model indication for MSC therapy down the line.

Results from clinical trials thus far have been varied. In order to gain a mechanistic understanding of the variability clinicians are observing, researchers at McGill University cross-examined MSCs from normal adult volunteers. Using an in vitro model, 7 different MSC lines were tested for their ability to suppress T cell proliferation. Results of this study indicate that MSCs possessing the most potent anti-immune power upregulate expression of an enzyme known as indoleamine 2,3-dioxygenase (IDO) in response to interferon-α. IDO is the first enzyme in the kynurenine pathway that catalyzes the degradation of trytophan. Several of the metabolites of this pathway are known to activate the stress response kinase CGN2, which causes apoptosis of T cells. Researchers also found that IDO activity is implicated in the differentiation of monocytes into immunosuppressive macrophages that regulate T cell proliferation in an IL-10 dependent fashion.

Elucidating the molecular mechanisms that contribute to the ability of MSCs to temper the immune system will allow us to generate MSC lines that have the most potent anti-immune power for the treatment of inflammatory and autoimmune disease.

Friday Science Review: December 30, 2011

Hedgehog Signaling Upholds Integrity of BBB

University of Montreal ♦ Published in Science, December 23, 2011

The blood-brain barrier (BBB) is a crucial boundary within the body that restricts the migration of blood-borne molecules and immune cells from circulation into the brain. Specialized endothelial cells tightly bound together with junctional proteins ensure that only certain small molecules are able to access the brain. Given that Hedgehog (Hh) signaling has been implicated in multiple sclerosis, a disease characterized by autoimmune destruction of myelin cells, researchers at the University of Montreal hypothesized that Hh signaling may have an influence on the formation of the BBB. They found that astrocytes in close proximity to endothelial cells in the BBB release Hh and that endothelial cells express Hh receptors, in keeping with the theory that Hh signaling is important for the integrity of the BBB. In order to demonstrate that Hh signaling also keeps leukocytes out of contact with the brain, researchers injected mice with cyclopamine, a small molecule inhibitor of the Hh pathway. Indeed, pharmacological inhibition of the Hh pathway led to acute disruption of the BBB and passage of leukocytes into the brain. Together these findings implicate the Hh pathway as being critical in the development and structural integrity of the BBB.

Friday Science Review: December 23, 2011

Super-Selective Oncolytic Virus

Ottawa Hospital Research Institute ♦ Published in Molecular Therapy (npg), December 20, 2011

More on oncolytic viruses this week but not with VSV this time, but rather the poxvirus JX-594. This particular virus, while having an excellent therapeutic index against multiple solid tumour types, is not that well understood. Researchers seeking to understand the mechanisms underlying its exquisite cancer cell selectivity identified multiple interactions. Three model systems were investigated, including primary normal and cancer cells, surgical explants, and mouse tumour models. It was found that selectivity of JX-594 was driven by multiple factors; the first time selectivity has been attributed to more than one specific mechanism. Among these factors were virus replication activation by the EGFR/Ras signaling pathway, cellular thymidine kinase levels (the virus is engineered to be responsive to TK so this was expected), and similar to VSV, hyporesponsiveness to type-I interferon. These finding will allow for the generation of more selective and potent oncolytic viruses.

Immunomodulators Enhance Polyfunctionality of T Cells Following Vaccination

McMaster Immunology Research Centre ♦ Published in Molecular Therapy (npg), December 20, 2011

Recombinant human adenovirus vaccines are capable of producing memory CD8+ T cell populations, however these populations lack polyfunctionality; in response to antigen stimulation they are not able to produce multiple different cytokines and chemokines. Researchers at McMaster have discovered that inhibiting mammalian target of rapamycin (mTOR) while stimulating OX40 alters not only the magnitude of T cell response, but also its phenotype and functionality. mTOR inhibition modulates differentiation of T cell pools while stimulation of OX40 enhances the process of costimulation. The addition of immunomodulators elicited greater immunity against multiple virus challenges in a mouse model, but was contingent upon sufficiently long transgene expression from the adenovirus under study.

Friday Science Review: December 16, 2011

Homeostasis in the Gut, Plasma Cells on Patrol

University of Toronto ♦  McGill University ♦ University of Bern ♦ Memorial University of Newfoundland

Published in Nature, December 11, 2011

The intestine is the largest mucosal surface in the body and is exposed to a diversity of microbes. This microbial life is healthy, although the immune system must still keep bacterial population sizes in check. One mechanism by which this is achieved is through the differentiation of B cells to plasma cells, which in turn secrete the immunoglobulin IgA. New findings indicate that plasma cells have additional roles that keep microbiota at bay. Researchers found that plasma cells secrete tumor-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), both of which exert antimicrobial activity. Microbial co-stimulation is pre-requisite for plasma cell multi-functionality. In order to investigate the effects that TNF-α and iNOS have on regulating flora in the gut, researchers used a double knockout mouse model. Removal of the two mediators led to a change in the composition of intestinal flora and a reduction in secretion of IgA from plasma cells.

VSV 2.0: IL-15 Engineered

McMaster University ♦ Published in Cancer Gene Therapy (npg), December 9, 2011

A popular approach to the development of oncolytic viruses for cancer therapy has been the use of the single-stranded RNA virus known vesicular stomatitis virus (VSV). This particular virus has a hypersensitivity to the protein type I interferon (IFN). Upon exposure to a virus the body releases IFN in attempt to prevent infection. The beauty of VSV is that while normal healthy cells are highly sensitive to IFN, tumour cells remain hyporesponsive. Systemic administration of VSV has little to no effect on normal cells as the virus cannot replicate within them. However, because they fail to respond to IFN, cancer cells remain susceptible to VSV infection, VSV replication, and eventually cell lysis. While VSV alone has been shown to be effective in preclinical models, long-term survival is not greatly increased.

Researchers at McMaster’s Centre for Gene Therapeutics have upped the anti with the development of a genetically engineered VSV that expresses an interleukin-15 (IL-15) transgene. IL-15 is a cytokine that has a critical role in adaptive immune responses to pathogenic entities. The group showed that injections of the IL-15 VSV in a mouse model of colon cancer led to strong localized expression of the cytokine in vivo. The new IL-15-rich microenvironment surrounding tumours increased the adaptive immune response targeted at tumour cells by recruiting and activating natural killer cells and T-cells. This novel virus has the two-fold effect of not only lysing cells, but enhancing anti-tumoral T-cell response. Mice receiving injections exhibited better survival overall.

Friday Science Review: December 9, 2011

hiPSC-derived HSCs Engraft Mouse Model

McMaster Stem Cell and Cancer Research Institute ♦ Published in Stem Cells, November 30, 2011

There are several sources currently being used to isolate hematopoietic stem cells (HSCs) for transplantation in humans. Included in these are the bone marrow, peripheral blood, and umbilical cord blood. However, HSCs derived from human embryonic stem cells (hESCs) have, as of yet, failed to be a viable source for transplantation. Although hESCs are capable of hematopoietic differentiation in the petri dish, their HSC progeny are unable to engraft human-mouse xenograft models. This investigation shows that human induced pluripotent stem cells (hiPSCs), which are also capable of hematopoietic differentiation in vitro, produce HSCs that can successfully engraft following transplantation into the mouse model; similar to HSCs from adult sources. This is an advance in terms of studying hematopoietic transplantation in humans from pluripotent sources. Despite the ability of hiPSC-derived HSCs to engraft they were unable to reconstitute hematopoiesis. However, they could establish colonies after being removed from the bone marrow compartment suggesting that a molecular mechanism blocks their capacity to differentiate in vivo. Investigators believe that misexpression of various microRNAs may explain why hiPSCs are unable to reconstitute hematopoiesis.

Breast Cancer Stem Cells, From Where Do They Originate?

British Columbia Cancer Agency ♦ University of Melbourne ♦ University of British Columbia

Published in Stem Cells, November 30, 2011

There has been a lot of talk recently about aldehyde dehydrogenase (ALDH) because it seems to be expressed across such a diversity of different stem cell types. We know that it is expressed by hematopoietic progenitors, mesenchymal progenitors, neural progenitors, and endothelial progenitors. Researchers investigating the expression of ALDH in a number of functionally defined mammary cell types have come across an interesting finding. They found that ALDH expression is actually lower in mammary stem/progenitor cells, that have bilineage differentiation potential, than it is in cells committed to particular lineages. Progenitor cells of the luminal lineage, for example, express relatively high levels of ALDH. It seems that a molecular switch upregulates ALDH, particularly the ALDH1A3 isoform, during the process of lineage commitment. The finding that ALDH is upregulated during differentiation challenges the notion that breast cancer stem cells arise from mammary stem cells. Recent reports have associated ALDH activity with breast cancer stem cells, but if ALDH expression is turned on during differentiation then breast cancer stem cells may in fact be the less primitive luminal progenitor. Further investigation of the temporal and spatial expression of ALDH in the breast should help explain this quandary.

Friday Science Review: November 25, 2011

Bacterial Response to Starvation Breeds Resilience

McGill University ♦ Published in Science, November 18, 2011

Biofilms are one of the primary mechanisms by which bacteria evade the toxic effect of antibiotics. Using a process known as quorum sensing bacteria can communicate amongst one another to accumulate in unison on a surface, living, synthetic, or natural. The benefits provided to the bacteria in film-formation are two-fold: firstly, the density of the bacteria reduces bacterial exposure to antibiotics, and secondly, a protective layer eventually encases the film as it exudes heavy polymeric substances.

However, there is a consequence to biofilm formation — nutrient deprivation and starvation. One would think this to be a disadvantage, but it actually further increases bacterial resistance to antibiotics. One hypothesis to explain this phenomenon is that starvation induces growth arrest and that this reduces the activity of factors that antibiotics require to kill bacteria. New findings from McGill indicate that there is more to the explanation than the passive resistance created through growth arrest. Researchers have discovered an active response to starvation, the starvation-signaling stringent response (SR), which increases tolerance to antibiotics. During times of starvation the SR mechanism is activated leading to a reduction in the burden of oxidants inside bacterial cells. A key finding in this study was that bacteria could be sensitized to antibiotics, by several orders of magnitude, by interfering with the SF mechanism.

Cystic fibrosis (CF) is a good example of a disease indication where biofilms contribute to morbidity. Aggressive Pseudomonas aeruginosa infections occur in the lungs and airways of CF patients. These infections can be treated during the early stages of the disease, however resistant biofilms lead to bacterial adaptation and chronic infection, which ultimately causes fatal complications. Future therapies for biofilms will likely involve a multi-faceted approach that target the many pathways and signaling mechanisms that lead to their formation.

Repression of Mitochondrial Translation: New Therapeutic Approach for AML

Ontario Cancer Institute ♦ Published in Cancer Cell, November 15, 2011

A chemical screen has identified the small molecule tigecycline as a potent suppressor of acute myeloid leukemia (AML). The screen involved 312 drugs that had already been approved by the FDA, both on-patent and off-patent, and focused on those that were well characterized antimicrobials or metabolic regulators. Tigecycline exhibited cytotoxic effects on two human AML cell lines, while having little effect on their normal hematopoietic counterparts. The molecule also had antileukemic activity in a mouse model of human leukemia. A genome-wide screen in yeast was carried out to elucidate tigecycline’s mechanism of action. The small molecule was found to inhibit a transcription factor. The interesting finding was that inhibition was not cytoplasmic, but instead mitochondrial. Tigecyline likely interacts with the EF-Tu transcription factor in the mitochondria, as researchers found they could mimic tigecycline’s effects by downregulating expression of EF-Tu using shRNAs.

Friday Science Review: November 18, 2011

NF-κβ1 Restrains Maturation of Dendritic Cells

Ontario Cancer Institute ♦ University of Toronto

Published in Nature Medicine, November 13, 2011

Dendritic cells (DCs) are critical for adaptive immune response, however if their proliferation becomes overzealous or their survival too prolonged they can cause autoimmunity. While most researchers have focused on the factors that stimulate DC maturation and autoimmunity, a group at the Ontario Cancer Institute has taken a different approach, and begged the question as to whether there exist factors that put a damper on DC maturation. Sure enough they recently discovered that NF-κβ1 maintains the resting state of DCs. Unstimulated DCs derived from mice lacking the Nfkb1 gene were able to activate CD8+ T lymphocytes. Injecting Nfkb1-/- DCs into mice could also produce diabetes, an autoimmune disease wherein the host’s immune system slowly eats away at insulin producing cells in the pancreas. NF-κβ1 ensures that DC production of TNF-α is regulated. Without NF-κβ1 the production of TNF-α becomes dysregulated leading to upregulation of granzyme B in T cells, which causes autoimmunity.

Human Spinal Cord-Derived NSPCs Survive Transplantation and Differentiate

Toronto Western Research Institute and Krembil Neuroscience Centre

Published in PLoS ONE, November 2, 2011

Researchers have provided a proof-of-principle that adult neural stem/progenitor cells (NSPCs) from the human spinal column can be successfully transplanted in a spinal cord injury (SCI) setting. The ability of NSPCs to engraft and differentiate in vivo was assessed with a mouse model of SCI. Following transplantation NSPCs differentiated into both neurons and glia. In order to establish NSPC lines, neural tissue samples were taken from the spine of organ transplant donors and grown in an adherent cell culture. NSPCs were selected and expanded in the presence of epidermal growth factor and fibroblast growth factor-2. Cultures can be maintained for at least 9 months and exhibit full multipotentiality in that they can differentiate into all three neural cells types, including neurons, oligodendrocytes, and astrocytes. In 2008, a research group successfully established an NSPC cell line, however was not able to scale the cells up for experimentation. This study is the first that shows NSPCs derived from the human spinal column can be scaled up for therapeutic purposes.

Friday Science Review: November 11, 2011

Genetically Modified MSCs for Acute Kidney Injury

McGill University ♦ Published in Molecular Therapy (npg), November issue

A research group has taken mesenchymal stem cells (MSCs) and enhanced them with a gene encoding erythropoietin (Epo) to show that they can be used to treat acute kidney injury (AKI). To generate a mouse model of AKI mice were injected with cisplatin, causing injury to the kidney. Epo-enhanced MSCs were then tested against unmodified MSCs for their capacity to regenerate tissue in the kidney and restore kidney function. Mice that received the Epo-enhanced MSCs exhibited improved survival and significantly better kidney function, as measured by blood levels of urea nitrogen, creatinine, and the enzymes amylase and alanine aminotransferase. Histological sections of recipient kidneys displayed less cell death and higher quantities of proliferating cells. Delivery of the Epo gene to modified MSCs causes the cells to secreate Epo protein, which is a known cytoprotective agent. This factor, in addition to the reparative effects of MSCs, produces a particularly strong regenerative effect upon transplantation.

Link Between p53 and Metabolism

York University ♦ University of Toronto ♦ Princess Margaret Hospital

Published in Molecular Cell, November 4, 2011

The tumour suppressor protein p53 has long been known to be the cell’s guardian. The protein can induce cell cycle arrest to allow for DNA repair, induce cellular senescence, or initiate signaling cascades leading to cell death if stress signals become overbearing. New findings suggest p53 is also involved in regulating metabolism on some level. A recent investigation has identified another link between p53 and metabolism, strengthening this hypothesis. Researchers found that the metabolism-regulating gene Lpin1 is partially under the control of p53. The lipin-1 protein controls the degree of fatty acid oxidation in the cell based on p53-signaling. Under normal glucose conditions, where cells can utilize glucose as a source of energy, the lipin-1 protein suppresses fatty acid oxidation. However, during times of nutritional stress lipin-1 promotes the oxidation of fatty acids to provide energy for cellular processes. In this study mouse myoblasts and human fibroblasts were cultured in conditions that induced nutritional stress. During nutrient deprivation reactive oxygen species (ROS) in the cell amass and a protein known as ATM kinase is activated, which in turn causes the activation of p53 and upregulation of Lpin1.

Friday Science Review: November 4, 2011

Native Microbiota Stave Off Infection

University of British Columbia ♦ Published in PLoS ONE, October 28, 2011

Genetics can contribute to host susceptibility to microbial infection but are there other causes? A growing body of evidence points to the composition of microbial flora in the intestine as a key factor in how mammals respond to foreign pathogens. In this study out of the University of British Columbia researchers investigated the effect that infection with a common pathogen had on a number of different strains of mice with widely varying genetic backgrounds. As would be expected the mice responded differently after oral exposure. Some mice were resistant to infection while others became very sick, or even died. However, it wasn’t only genetics at work. Transplantation of microbial samples from the intestines of resistant mice into those of lethally susceptible mice prolonged microbial colonization and death, illustrating the importance that the microbial ecosystem has in regulating mucosal immunity and intestinal health.

Selection of Cardiomyocytes with SIRPA

McEwen Centre for Regenerative Medicine (UHN) ♦ University of Toronto ♦ Monash University

Published in Nature Biotechnology, October 23, 2011

Gordon Keller’s lab has discovered a protein, known as signal-regulatory protein alpha (SIRPA), that can enrich for embryonic stem cell and iPS cell-derived cardiomyocytes at purities as high as 98%. Keller, who was the first researcher to produce functional heart cells from human embryonic stem (hES) cells, has spent years investigating the developmental path that leads to the cardiac identity. In order to find this specialized protein marker researchers carried out a screen of cardiomyocytes against a panel of some 380 CD antibodies. SIRPA was found to be expressed specifically on cardiomyocytes that were the progeny of hES cells or iPS cells; the protein will allow for rapid and efficient separation of cardiomyocytes from both primitive cell types.

Engineering Therapeutic Viruses for Greater Impact

Children’s Hospital of Eastern Ontario Research Institute ♦ University of Ottawa ♦ Ottawa Hospital Research Institute ♦ OICR ♦ McGill

Published in Cancer Cell, October 18, 2011

Viruses are now being leveraged in order to target and destroy cancer cells in the body. Termed “oncolytic” viruses, these treatments not only infect and burst cancer cells but also alert the host’s immune system to the presence of the tumour ensuring a continued onslaught following administration. In hopes of improving upon oncolytic viruses currently under development, researchers have implemented functional genomics to glean information related to virus-tumour interactions. A genome-wide RNAi screen identified the endoplasmic reticulum (ER) stress response as a mechanism by which tumour cells resist the effects imposed by oncolytic viruses. Inhibiting the ER stress response pathway sensitized resistant tumour cells and increased the efficacy of oncolytic viruses by up to 10,000 fold in some cases.

Friday Science Review: October 21, 2011

CD34: Beyond A Stem Cell Marker

Biomedical Research Centre, Vancouver ♦ University of British Columbia ♦ Sprott Centre for Stem Cell Research, Ottawa ♦ Others..

Published in Stem Cells, October 13, 2011

Despite the fact that the cell surface marker CD34 is typically used to identify a diversity of adult stem cell types, no regeneration-related function has been attributed to it. In attempts to broaden our understanding of the protein’s contribution to regenerative biological processes, researchers at the University of British Columbia probed the effect that it has on muscle regeneration in mice. Knockout mice lacking the protein still developed properly, however exhibited an inability to regenerate muscle following both acute and chronic skeletal muscle injury. Researchers attribute this defect to a shortcoming in satellite cells; a small and rare progenitor cell type found in muscle that migrates to the site of injury to proliferate and restore muscle mass. In the absence of CD34, it appears that satellite cells are unable to migrate along muscle strands to relocate themselves and stimulate the growth of new tissue.

Black Death Reconstructed

McMaster University ♦ University of Tübingen ♦ Max Planck Institute of Evolutionary Anthropology ♦ University of South Carolina ♦ Others..

Published in Nature, October 12, 2011

Scientists working out of McMaster University’s Ancient DNA Centre have managed to reconstruct the genome of Yersinia pestis. The rod-shaped microbe was responsible for the epidemic that killed an estimated 30-50% of the European population between the years 1347 and 1351; the infection was appropriately given the name the Black Death. The advent of high-powered sequencing technologies has allowed us to quickly and accurately map the genomes of ancestral microbes. Not only do these genomes provide valuable insight into their biology, they allow us to plot the evolution of pathogens and adaptations they have gathered over time. DNA samples were collected from the teeth of skeletons unearthed from old burial grounds in London. Contrary to what was originally believed, the sequenced genome revealed that the ancestral microbe was no more virulent than strains in existence today. Researchers believe the severity of the 14th century pandemic was brought on by the generally malnourished and immuno-compromised state of the population at that time. Poor hygiene and particularly wet conditions are likely to have exacerbated the spread of the microbe as well.

Friday Science Review: October 14, 2011

Ubiquitin Ligase Sweeps Aggregates

University of British Columbia ♦ Published in Nature Cell Biology, October 9, 2011

It seems that ubiquitin ligase may play a critical role in preventing diseases characterized by protein aggregation. The enzyme acts by fusing a polyubiquitin chain to misfolded proteins that sit in the cytoplasm. Once tagged, these faulty proteins are degraded by other enzymes in the proteasome. Researchers at the University of British Columbia utilized heat-shock to create short-term stores of misfolded proteins in yeast cells and demonstrated that a specific ubiquitin ligase, Hul5, is responsible for ubiquitylation and removal of low-solubility proteins. Pin2, a protein involved in the development of prion disease, was also observed to be removed by Hul5.

Key Determinant of Osteoporosis Discovered

University of Montreal ♦ McGill University ♦ Laval University ♦ Washington University School of Medicine

Published in Cell Metabolism, October 5, 2011

Researchers have discovered a factor that controls the differentiation of osteoclasts, the cell type responsible for bone resorption. Bone resorption is one of two processes that act in concert to ensure a constant bone mass in the human body. When this balance is broken the result is loss of bone density, otherwise known as osteoporosis. When resorption occurs osteoclasts break down releasing calcium into the bloodstream which in turn regulates bone formation. Inppb4α seems to regulate osteoclastogenesis by repressing the differentiation of osteoclasts. Mice deficient in Inppb4α exhibit accelerated osteoclast differentiation and bone loss. An important finding in this study was that the human gene, INPP4B, was found to be a susceptibility locus for osteoporosis, confirming the functional analysis carried out in mice.

Antisense Oligos for Huntington Disease

University of British Columbia ♦ Isis Pharmaceuticals ♦ University of Copenhagen

Published in Molecular Therapy, October 4, 2011

Huntington disease (HD) is characterized by a CAG nucleotide expansion in the Huntington gene (HTT). Hence, targeting the HTT gene has been a desirable therapeutic approach. However, the wild-type (normal and healthy) HTT gene is crucial for neuronal development and survival so down regulating its expression can have deleterious effects. To circumvent this researchers created gene-silencing antisense oligonucleotides (ASOs) that target small variable regions known as single nucleotide polymorphisms (SNPs) found in the HD population. ASOs were modified with S-constrained ethyl (cET) motifs to ensure selectivity of target sites. Delivery of ASOs to the mouse brain demonstrated allele-specific knockdown of the HTT protein in vivo.

Optimizing Lipid Nanoparticles for Delivery of Therapeutic Payload

University of British Columbia ♦ Published in Molecular Therapy, October 4, 2011

One of the most effective means to deliver therapeutic payloads is to drop them into lipid nanoparticles (LNPs) for in vivo delivery. LNPs are easier to manufacture than viral vectors and have lower immunogenicity, making LNP delivery systems an attractive option. In this study researchers investigated the potency of RNAi on primary bone macrophages and dendritic cells using any one of four cationic lipids to build LNPs. Of the four lipids used, DLinkC2-DMA provided the most potent intracellular delivery in vitro and in vivo. To demonstrate the efficacy of DLinkC2-DMA-containing LNPs researchers loaded them with GAPDH-siRNA and administered them intravenously to mice. Significant inhibition of GAPDH and CD45 was observed in spleen and peritoneal macrophages and dendritic cells in relation to LNPs containing other lipid types.

Long Non-Coding RNAs, Dark Matter Comes into Light

British Columbia Cancer Agency Research Centre ♦ University of British Columbia

Published in PLoS ONE, October 3, 2011

Researchers have had a change of heart with respect to the contribution that long non-coding RNAs (LncRNAs) have to cancer formation. LncRNAs do not code for proteins, instead serving structural and functional roles in the cell. Due to the fact that they are not actually translated to proteins, LncRNAs have been dubbed the ‘dark matter’ of the transcriptome. Conservative estimates put their numbers somewhere in the vicinity of 23,000 transcripts. LncRNAs have been implicated in at least two cancer types, breast cancer and lung cancer, but this paper is the first large scale analysis of their contribution to human cancers. By comparing 26 different normal human tissues and 19 human cancers, researchers show that in many cancers LncRNAs are aberrantly expressed and may not be as innocuous as most think. Today’s current commercial microarrays used for transcriptome profiling focus on coding regions of the transcriptome. We may now have to place more emphasis on non-coding regions in order to capture a more comprehensive view of how the transcriptome can contribute to the development of cancer.

Friday Science Review: October 7, 2011

Cell Polarity Dictated by Phosphatidylserine

Hospital for Sick Children ♦ Published in Nature Cell Biology, October 2, 2011

The enzyme Cdc42 is responsible in part for cell polarization during asymmetric cellular events. In the case of the yeast S. cerivisiae, polarization is essential during the process of budding and the formation of projections in response to mating factors. Polarization is also crucial to the operation of mammalian cells. In this Nature paper researchers show that phosphatidylserine, a type of phospholipid, is required for the correct dispersal of Cdc42 during polarization events. In an event that precedes the movement of Cdc42, phosphatidylserine is sent via secretory vesicles to the plasma membrane where it accumulates. Yeast mutants lacking phosphatidylserine synthase exhibited impairment in the polarization of Cdc42 and difficulty in budding and mating, suggesting it is at the core of regulating cellular polarization and some very important cellular processes.

Embryonic Stem Cell-Specific Splicing Event Promotes Pluripotency

University of Toronto ♦ Mount Sinai Hospital ♦ Columbia University Medical Centre ♦ Published in Cell, September 30, 2011

There are a host of genes that are upregulated in embryonic stem cells which promote stem cell-like characteristics, namely pluripotency, or the ability to give rise to all cell types in the human body. These genes include Sox2, Oct4, and Nanog, among others, and are associated with primitive cell types. Much time has been invested in researching the transcriptional networks promoting pluripotency, and how the various stem cell-specific transcription factors cross-talk with one another to ensure stem cells do not differentiate. Although these transcription factors can cross-regulate each other, on a more fundamental level they are controlled by the forkhead box (FOX) transcription factors. In this study, researchers discovered that FOX transcription factors do not always operate in the same manner. In stem cells, the FOXP1 mRNA transcript is alternatively spliced in such a manner that the FOXP1 protein has a greater affinity for the DNA promoter regions that control transcription of stem cell factors like Sox2, Oct4, and Nanog. FOXP1 also represses genes that cause differentiation. Interestingly, the FOXP1 isoform studied here could not be found in more differentiated cells, suggesting that alternative splicing is an important mechanism by which stem cells maintain their primitive status.


Friday Science Review: September 30, 2011

The Dynamic Immunopeptidome

University of Montreal ♦ Published in Molecular Systems Biology, September 27, 2011

The human body has developed a complex system in which to identify self and non-self. The large majority of nucleated cells in the body display small peptides on the cell surface known as MHC I-associated peptides (MIPs). Although past theories have been conducive to the view that the creation and expression of these peptides remains the same regardless of cellular conditions, researchers now believe that the pattern of MIPs presented on a cell may in fact be dynamic and change according to metabolic conditions. Taking a high-throughput mass spectrometry approach, researchers investigated changes that occurred in the immunopeptidome of a mouse lymphoma cell line. In order to alter metabolism, cells in the treatment group were exposed to rapamycin, a potent inhibitor of the kinase mTOR which is known to regulate cellular homeostasis. Rapamycin induced wide-sweeping changes in the MIP composition on the cell surface. A major finding in this study is that the immunopeptidome displayed on the cell surface actually provides a representation of the various biological events occurring within the cell.

Calcium Channel Key to T Cell Development

University of British Columbia ♦ University of Calgary ♦ Oxford University ♦ Published in Immunity, September 23, 2011

Calcium channels are critical players in physiology, development, and cell metabolism. Researchers have identified a specific calcium channel, CaV1.4, that is essential for channeling calcium ions into the cytoplasm of naive T cells. Intracellular calcium stores control signaling, differentiation, and activation. Researchers also found that CaV1.4 is in part responsible for modulating T cell receptor induced rises in calcium stores. In order to investigate the effects that CaV1.4 has on cell homeostasis researchers used a method known as patch clamp. This technique can record the conductance of single cells. Measurements of thymocytes and peripheral T cells from mice deficient in the CaV1.4 channel indicate that the channel both increases intracellular calcium stores on its own and affects the ability of T cell receptors to elevate intracellular calcium stores. Findings also indicate that CaV1.4 may to a certain degree control survival of developing T cells.

Also see this review published in Trends in Biotechnology, on recent methods used to understand host-virus interactions and identify novel targets for diagnostic and therapeutic applications.

Friday Science Review: September 23, 2011

Gene Therapy Tailored to the Prostate

University of British Columbia ♦ Published in Cancer Gene Therapy (npg), September 16, 2011

Prostate cancer cells tend to over express the protein translation initiation factor 4E (eIF4E). With this knowledge on hand, researchers at the University of British Columbia experimented with a variety of 5′UTRs to see which was most sensitive to eIF4E, and hence, which would drive the most efficient gene transfer for treating prostate cancer. The untranslated region (UTR) of mRNA is a non-coding portion of RNA upstream of the coding region which provides a docking site for translational machinery during protein synthesis. Researchers investigated the 5′UTR of fibroblast growth factor-2 (FGF-2) and another two from ornithine decarboxylase. It was determined that the 5′UTR of FGF-2 provides the best translational efficiency, as measured by a GFP reporter. Findings were confirmed by injecting viral vectors containing the three 5′UTRs into the prostates of PTEN deficient (tumour bearing) mice.

Mechanism of Post-Stroke Immune Suppression Elucidated

University of Calgary ♦ Published in Sciencexpress, September 15, 2011

One of the consequences of stroke is severe inflammation of the brain. As a result, the body has evolved an immune suppression mechanism to compensate for this swelling to avoid brain damage. The exact nature of this mechanism remains unknown. It seems that immunomodulation of natural killer T (NKT) cells may be one avenue that the body takes to avoid inflammation. Using a mouse model of stroke researchers recently showed that the nervous system suppresses activation of NKT cells by altering its innovation on the liver, the organ in which NKT cells primarily reside. Immune suppression following stroke is problematic as it can lead to infection, especially in the elderly, where the immune system is already somewhat diminished. A better understanding of immunomodulation following stroke should aid in the development of drugs that support the immune system post-stroke.

Excisable Marker for Creation of Pox Vectors

University of Ottawa ♦ University of Alberta ♦ Published in PLoS ONE, September 8, 2011

Gene transfer requires effective vectors in which to carry genetic material. Viruses are often exploited to produce DNA vectors, where their genomes are modified through the addition of foreign DNA of therapeutic value. The process of creating viruses for gene transfer begins with the introduction of foreign DNA to a packaging cell type using circular pieces of DNA known as plasmids. Once inside the cell, these plasmids are taken up by the genome of the packaging cell. The cells are then used as factories that produce viral particles containing therapeutic DNA. For the process to be efficient, one must be able to detect cells that have been successfully transformed with plasmid DNA. Reporters such as green fluorescent protein or luciferase or often used, but by incorporating them into plasmids non-therapeutic DNA is left behind that serves no purpose. Researchers have circumvented this issue by creating plasmids containing an excisable marker element. These plasmids were used to create marker free recombinant poxviruses. To allow for removal of the reporter, specific sequences of DNA known as lox sequences were placed on either side of it. The Cre/Lox system was then used to excise the marker prior to the production of viruses. Viruses for gene transfer without marker elements are considered more therapeutically relevant, as they do not contain extraneous traces of DNA.





Friday Science Review: September 16, 2011

Novel Genetically Encoded Calcium Indicators

University of Alberta ♦ Hokkaido University ♦ Kyushu University

Published in Science, September 8, 2011

Calcium transport is critical to normal physiology having an essential role in processes like neural communication and muscle contraction. As a result, the element has been at the centre of a large body of physiological research. Over the years researchers have attempted to trace calcium to monitor physiological reactions, such as neurotransmitter release from neurons or the contraction of cardiac cells. Fluorescent indicator proteins are one means in which to do this. When calcium is present in a system that contains a fluorescent indicator it becomes bound to the indicator causing it to emit a characteristic energy that can be observed using fluorescence microscopy. Although this technology has been around for decades, continuing advances in microscopy have placed new fluorescent indicators in demand. Researchers at the University of Alberta have constructed not just one, but a cassette, of calcium reporters for research use. This cassette is composed of indicators that fluoresce in one of three colours — blue, green, or red; a step up from previous indicators that could only weakly fluoresce in a green hue. These novel proteins are expected to advance our understanding of calcium’s contribution to physiology and allow researchers to complete experiments that were previously impractical to approach.

At the Interface

Holland Bloorview Kids Rehabilitation Hospital ♦ University of Toronto ♦ University of Pittsburgh ♦ Hospital for Sick Children

Published in PLoS ONE, September 7, 2011

Brain computer interface (BCI) technology may one day revolutionize the way humans control devices. Researchers are currently investigating BCIs as a means to translate mental thought to signals that can control external devices. If commercialized, the technology could be useful in areas such as virtual reality, wheelchair control, and speech in individuals that lack muscle control. Several different modalities have been developed to register thought, including electroencephalography, functional magnetic resonance imaging, and magnetoencephalography. However, these methods still have shortcomings that limit their practicality. A technique known as transcranial Doppler ultrasound (TCD), which measures cerebral blood flow velocity in the brain, has shown promise as a new paradigm for BCI systems. Using TCD, researchers were able to measure changes in the velocity of cerebral blood flow in response to a word generation task and a mental rotation task. The system was able to decipher between the two with high accuracy suggesting that TCD could one day be useful for the more complex tasks that BCI will demand.

Also checkout this review on the use of encapsulation technology for applying biofertilizers and biocontrol agents, and this review on extracting polyphenols from plants for analysis in the lab; both papers published in Critical Reviews in Biotechnology.

Friday Science Review: September 9, 2011

Astrocytes Modulate Basal Synaptic Transmission

University of Montreal ♦ Montreal General Hospital ♦ Published in Cell, September 2, 2011

The astrocyte, a larger flattened cell type relative to the neuron, regulates the growth and survival of brain cells through the release of various growth factors and proteins. Recent findings indicate that they are also involved in regulating basal synaptic transmission. Although it has been known for some time that astrocytes are involved in modulating sustained synaptic transmission in the brain, researchers are in less agreement over whether or not astrocytes modulate basal synaptic transmission involving single synapses.  In this study, researchers found that astrocytes were activated by single action potentials and responded to this activation by releasing purines, specifically adenosine, which in turn increased basal transmission. It is believed that astrocyte activation occurs by way of the metabotropic glutamate subtype 5 receptors found on certain functional areas of the astrocyte process. After being release, purines bind to presynaptic A2a receptors on neurons, heightening basal synaptic transmission.

Neurogenesis Regulated by Alternative Splicing

University of Toronto ♦ Hospital for Sick Children ♦ King’s College London ♦ Published in Molecular Cell, September 2, 2011

Researchers have discovered a key mechanism that regulates the complex process of neurogenesis. Like many complex processes in the body, the process of neural development is controlled by a large array of genes and regulatory pathways. It appears that neurogenesis is activated by a protein known as nSR100, which alternatively splices another gene REST. Alternative splicing occurs when a gene is not transcribed by the cell’s protein machinery in its regular form. Instead, an alternative form, slightly different than the regular form (known as an isoform), is produced. This isoform can have reduced function or lose function entirely. In the case of REST, its alternative splice isoform REST4 still functions, however has vastly reduced activity. REST typically negatively regulates a panel of genes that promote neurogenesis, so in the presence of the isoform neurogenenesis increases. Experiments with mice confirm these findings; blocking nSR100 in the mouse brain impairs neurogenesis.

Maternal RNA Transcript Epigenetically Regulates Gene Expression

University of Toronto ♦ Published in Science, September 2, 2011

RNA can function in a number of ways above and beyond acting as a messenger between DNA and the translational machinery found in the cytoplasm. Researchers studying sex determination in C. elegans recently showed that expression of a wild-type allele controlling sex-determination, fem-1, can be regulated by a maternal fem-1 RNA transcript in the germ line. In C. elegans, the nematode worm frequently used as a model organism in studies of genetics, expression of the fem-1 gene is required for male development. In this study, females engineered to express deletions of the fem-1 gene at both alleles gave rise to offspring that exhibited a strong bias towards the female sex in their germ line. Researchers were able to rescue this defect in progeny by injecting a non-coding fem-1 RNA into the maternal germ line. Because the defect is heritable, scientists postulate that fem-1 is typically repressed epigenetically, and that a maternal fem-1 RNA transcript acts to block repression of the wild-type fem-1 gene allowing males to be produced under normal circumstances.

Friday Science Review: September 2, 2011

Functional Validation of Cancer Stem Cell Theory

Ontario Cancer Institute ♦ University of Toronto ♦ Hospital for Sick Children ♦ McMaster University ♦ others..

Published in Nature Medicine, August 28th, 2011

John Dick’s lab has begun functionally validating the cancer stem cell theory in a mouse model and created the closest thing we have to a true leukemia stem cell (LSC) signature. The cancer stem cell theory operates under the premise that not all cells in a tumour can support malignant growth. Instead, only a small, and rare subset of cells at the top of a complex hierarchy is able to sustain the life of a tumour.

Researchers initially attempted to identify cancer stem cells through transplantation into xenograft models. Those fractions that could promote and sustain tumour growth were isolated and analyzed in order to create a cancer stem cell fingerprint. However, it has been discovered that the mouse xenograft models used to create these fingerprints were not sensitive enough. As a result, some cancer stem cells have gone unnoticed.

In order to ensure that the CSC characteristics identified through these experiments are clinically meaningful, they must be linked to patient outcome and survival. This is the first study to take such a robust approach to confirming CSC function, setting a precedent for future experiments in the CSC field. To illustrate how this can be achieved, researchers analyzed fractions of cells from acute myeloid leukemia (AML) tumours from 16 different patients.

The process began by creating four populations of AML cells using the major LSC surface markers (CD34, CD38). These populations were functionally validated through transplantation into a very sensitive xenograft mouse model. Functionally defined LSCs (tumour forming) were subsequently analyzed using global gene expression analysis. Bioinformatic analysis was then utilized to compare 25 LSC-enriched fractions to 29 fractions without LSCs.

Despite the fact that AML samples exhibited different karyotypes and patients were of varying backgrounds (French, British, American), this comparison generated a common LSC signature composed of 42 genes. This signature was then compared to a hematopoietic stem cell (HSC) signature derived from cord blood by similar functional analysis. As could be expected, the genetic profile of LSCs derived from the AML cell lines was quite similar to that of the HSC profile derived from cord blood stem cells; the differences in the LSC and HSC signatures representing genetic targets for treating AML.

The true testament to the value of this study is illustrated by the tests for clinical relevance that followed the formation of LSC and HSC signatures. Correlations between the LSC/HSC signatures and clinical outcome were evaluated using three comprehensive, clinically annotated, gene expression sets. Researchers found a significant negative correlation between complete remission and overall survival, and high expression of the LSR and HSC signatures.

This eloquent work by John Dick’s lab shows us that strong connections can be made between CSC signatures and clinical outcome. Researchers studying other cancer stem cell types should use this study as a model in which to investigate their identity and clinical relevance.

Friday Science Review: August 19, 2011

Host-Cell Nucleolin, A Helping Hand for RSV

University of British Columbia ♦ University of Toronto ♦ Published in Nature Medicine, August 14th, 2011

Human respiratory syncytial virus (RSV), a Pneumovirus that is widely responsible for respiratory tract infections worldwide, appears to use host-cell nucleolin for viral entry. After making the discovery, researchers corroborated their finding with several experiments to show the importance that nucleolin plays in RSV infection. Neutralization experiments with nucleolin antibodies in vitro decreased RSV infection, while competition experiments with soluble nucleolin placed in cell culture medium also inhibited the virus from passing into cells. Interestingly, insect cells that are typically immune to the virus could be made susceptible through the transfection of human nucleolin. Mice given a prophylactic dose of RNAi, knocking down nucleolin in the lung, exhibited significant reduction in RSV infection. RSV interacts with host-cell nucleolin with a glycoprotein found in the viral envelope. There is currently no effective therapeutic or vaccination for the virus. Palivizumab (manufactured by MedImmune) is available for patients considered to be at high risk, but remains expensive.

Calpain-Mediated Degradation Spurs Autoimmune Disease

University of Toronto ♦ Samuel Lunenfeld Research Institute ♦ Toronto General Research Institute ♦ Princess Margaret Hospital ♦ Others..

Published in Nature Genetics, August 14th, 2011

The human Lyp protein tyrosine phosphatase (Lyp620W) plays a critical role in immune homeostasis by regulating T-cell antigen receptor signaling. A mutant of Lyp620W, Pep619W, is associated with increased risk of autoimmune disease. The mechanism by which this mutation promotes autoimmune disease is largely unknown. Recent findings suggest that degradation of Lyp protein is at the root of immune dysfunction. When analyzing Lyp transcripts in both wild-type mice and mice containing mutant alleles of the gene encoding Lyp620W, researchers found that transcript levels remained the same despite mutation. However, in the case of mice homozygous for the mutant allele, there was a drastic reduction in Pep619W protein. It appears that the enzyme calpain-1 degrades mutant versions of the protein, preventing Lyp protein from regulating the immune system. In the absence of functioning Lyp620W, lymphocyte and dendritic cell hyperresponsiveness ensues, leading to deleterious disease conditions.

Also see this review (written by U of T researchers and published in Trends in Biotechnology) on the use of biomaterials to create supportive micro-environments for the transplantation of neural stem/progenitor cells into the central nervous system..

Friday Science Review: August 12, 2011

Acquisition of Animal Gene by Horizontal Transfer

University of Ottawa ♦ University of British Columbia ♦ University of Illinois ♦ Albert Einstein College of Medicine

Published in Current Biology (Cell Press), August 9, 2011

Researchers have recently made a rather surprising discovery — an animal gene that appears to have been taken up by the microsporidian Encephalitozoon romaleae. This is the first time that an animal gene has been documented in a microsporidian genome. Microsporidians often parasitise arthropods, and have been known to take up foreign DNA from bacteria through a process known as horizontal gene transfer. New DNA allows microsporidians to gain new functions that promote survival. After carrying out a screen of the microsporidian genome they found but one candidate animal gene, which was identified as a purine nucleotide phosphorylase (PNP). Phylogenetic analysis later revealed that the gene was likely of arthropod origin and could have been taken up by E. romaleae after it parasitised an insect.

Genetics Underpinning ALS

University of Montreal ♦ Published in PLoS Genetics, August 4, 2011

Using the zebrafish as a model, researchers have elucidated the connection between some of the common mutations associated with amyotrophic lateral sclerosis (ALS). Mutations in the genes SOD1 and TARDBP have been known to cause ALS, as has a mutation in the Fused in sarcoma gene (FUS). In this study researchers show that mutations in the FUS gene cause a loss of function; they cannot rescue the motor phenotype caused by a knockdown of the zebrafish Fus gene. It seems that the FUS and TARDBP mutations share a common pathogenic mechanism, as there co-expression did not exacerbate the phenotype observed when either were expressed alone. Findings indicate that SOD1 works through a second distinct mechanism however, as wildtype SOD1 was unable to rescue the phenotype observed after overexpression of FUS and TARDBP mutations.

Balancing Host Defense and Wound Repair

University of Toronto ♦ Hospital for Sick Children ♦ Benaroya Research Institute ♦ University Hospital Erlangen

Published in Immunity (Cell Press), August 3, 2011

Tak Mak’s lab provides some new insights into the mechanism by which thymic stromal lymphopoietin (TSLP) modulates the immune system in order to curb inflammation and launch the healing process in the colon. Using a knockout mouse model researchers illustrate that the absence of TSLP does not increase the severity of colitis, but instead prevents healing and recovery from the disease. Mice without the TSLP gene exhibited increased concentrations of an enzyme known as neutrophil elastase and decreased expression of secretory leukocyte peptidase inhibitor (SLPI). Typically SLPI serves to stomp out some of the immune response following insult to the colon by acting as a serine protease inhibitor and inhibitor of NF-κB. In the absence of TLSP signaling, however, it seems SLPI is downregulated, preventing mucosal healing.

Friday Science Review: August 5, 2011

High-Throughput Platform for Single-Cell qPCR

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

Published in PNAS, August 1st, 2011

An achievement in microfluidics this week as researchers from the University of British Columbia report on the development of an integrated microfluidics device that performs high-throughput mRNA analysis on a chip. The platform is capable of analyzing hundreds of single cells per run, and operates on a nanolitre scale, offering very high sensitivity. The goal of advanced microfluidics systems for single cell analysis has been to incorporate each step of the process into seamless automation. Although current devices provide the necessary high-throughput qPCR readout, they require micropipettes or cell-sorting to trap cells for analysis on the front-end. This novel device does it all, and is the first example of a fully integrated platform for single-cell qPCR on a chip. Among the other advantages of this platform are improved performance, reduced cost, and scalability.

Melarsoprol Exacerbates HIV-1 Infection

Laval University ♦ Sherbrooke University ♦ Published in Journal of Molecular Biology, July 29th, 2011

Recent findings suggest that melarsoprol, a drug commonly used to treat sleeping sickness in Africa, improves the HIV virus’ ability to replicate in human cells. Due to the fact that Trypanosoma brucei infections are often seen along with HIV-1 infection, researchers were curious to investigate potential interactions between the two pathogens. In doing so they found that melarsoprol can stimulate the replication of several strains of HIV-1 in dendritic cells. These findings raise concerns for patients receiving the drug because it can exacerbate disease conditions and may have implications on disease transmission. Melarsoprol seems to boost HIV replication by inhibiting a certain cellular restriction factor, which under normal circumstances exerts a slightly antagonistic effect on the HIV reverse transcription process.

Recombinant BCL-X Enhances Pre-Implantation Embryo Development

University of Toronto ♦ Mount Sinai Hospital ♦ Washington University in St. Louis

Published in PLoS ONE, July 20th, 2011

More automation this week with the introduction of a fully-automated robotic system for high-throughput microinjection of pre-implantation embryos. Infertility is on the rise, and as more and more couples require the services of IVF doctors, there is more demand for treatment options that can continue to increase the success of IVF in the clinic. This new system combines motion control, computer vision microscopy, and micro device technology to accurately and efficiently deliver small quantities of recombinant material. Researchers injected the anti-apoptic agent BCL-X into mouse zygotes, and found that they could enhance the viability and pre-implantation development of mouse embryos grown in sub-optimal in vitro conditions. The system could offer a new treatment paradigm for couples experiencing difficulty with IVF.

Friday Science Review: July 29, 2011

TK/GCV Suicide Gene Therapy: Connecting the Dots

Laval University ♦ Published in Cancer Gene Therapy (npg), July 22, 2011

Glioblastoma is an aggressive form of brain cancer requiring intensive therapy. Even with surgery, chemotherapy, and radiation, the mean survival time is approximately one year. A new therapeutic paradigm is currently being investigated in clinical trials, wherein a lentivirus delivers the herpes simplex virus thymidine kinase (TK) gene to the tumour bed following surgery. Tumour cells that are infected with the TK gene produce the TK protein, which in turn sensitizes tumour cells to the chemotherapeutic ganciclovir. After ganciclovir enters tumour cells it is phosphorylated by TK and then broken down into various toxic compounds that lead to cell death. In a nutshell the cell is altered genetically such that it commits suicide in the presence of the chemotherapeutic. One aspect critical to the success of this approach, however, is the degree to which tumour cells are connected; the therapy relies on something known as the “bystander effect”.

Researchers at the University of Laval have recently completed a comprehensive investigation on gap junctions in human glioblastomas. Gap junctions form between cells to allow intercellular communication through the migration of small molecules and peptides. They can be leveraged, therapeutically speaking, to amplify the efficiency of cancer therapies because they connect multiple cells into larger units. In the case of the suicide gene therapy described above, TK protein can migrate from cells that were successfully infected with the lentivirus into untransformed cells such that they are also eradicated in the presence of ganciclovir. In this study researchers found that connexin 43 (Cx43), the unit that assembles to form gap junctions in astrocytes, has heterogenous expression across glioblastoma cell lines. Analysis of 74 glioblastoma cell lines shows that Cx43 expression can be unaltered, reduced, or lost entirely. Cx43 expression was observed in 77% of cell lines, and its presence was adequate to provide a bystander effect regardless of whether its localization was considered normal or aberrant. Previous studies of smaller sets of glioblastoma cell lines had contradicting results, with some linking disease progression to loss of Cx43, while others supported the notion that high levels of Cx43 expression exacerbate disease conditions by promoting tumour cell infiltration into healthy tissue. This study provides the first comprehensive look at Cx43 in glioblastoma and should provide valuable information to companies in industry developing therapies that require a bystander effect for their success.

Structure of AMA1/RON2 Complex Elucidated

University of Victoria ♦ Published in Science, July 22, 2011

Apicomplexan parasites, including the Plasmodium species that cause malaria, have a very interesting mechanism that allows for host cell invasion. Upon contact with a host cell, these parasites release a structure known as a rhoptry neck (RON) complex that implants itself in the host cell membrane. A domain on RON2 then acts as a binding site for the apical membrane antigen 1 (AMA 1) displayed on the parasites cell surface. Essentially, apicomplexan parasites have evolved a means to provide both receptor and ligand to dock themselves on host cells. Once formed, the complex organizes itself into a structure known as a moving junction (MJ), a ring-like structure deeply embedded in the host cell membrane.  As this structure migrates from the anterior to the posterior of the parasite, the parasite becomes enveloped in a vacuole that is then internalized into the cell. Now researchers have elucidated the structure of the MJ complex at 1.95 angstrom resolution, providing valuable insight into its machinery. It is hoped that the structure can be used to develop therapies that prevent host cell invasion by apicomplexan parasites.

Friday Science Review: July 22, 2011

Genetic Basis for Gray Platelet Syndrome

Hospital for Sick Children ♦ University of Toronto ♦ University of Colorado ♦ University of Utah ♦ Others..

Published in Nature Genetics, July 17, 2011

Researchers have found a mutation in the gene NBEAL2 which seems to be at the root of Gray Platelet Syndrome (GPS), a disorder characterized by thrombocytopenia and enlarged platelets that lack α-granules. The discovery was enabled by RNA sequence analysis of platelets taken from an individual with autosomal recessive GPS. The mutation prevents recognition of a splice site at exon 9 in the mRNA strand encoding the protein neurobeachin-2. As a result of intron retention, premature stop codons are introduced to the mRNA strand; stop codons are known to cause mRNA degradation or protein truncation. Neurobeachin-2 contains a BEACH domain that is thought to be involved in cellular trafficking, and a defect in this protein likely leads to the manifestation of GPS.

Cell Fate Potential is Primed by Histones

McMaster University ♦ Published in Cell Stem Cell, July 8, 2011

Mick Bhatia’s lab has dug a little deeper into the mechanisms that bias lineage specification and differentiation of human embryonic stem cells (hESCs). Historically, it has been assumed that human embryonic stem cells are “equipotent”, meaning each and every one has the same potential, or equal potential, to differentiate into any of the more than 225 cell types in the human body. This theory has evolved over the last few years, and these recent findings confirm that the scene is quite a bit more colourful than originally thought. Embryonic stem cells are in fact restricted to certain lineages and this restriction is encoded by histone modification and patterning. Researchers in Bhatia’s lab illustrate this by fractionating stem cells in culture based on two surface markers, c-KIT and A2B5, and predicting cell fate based on histone modification marks observed on gene loci associated with pluripotency or various lineages.

Sorting with CD49f Provides Highest Reported Purity of HSCs

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

Published in Science ♦ July 8, 2011

Purification of specific cell types is essential to their study in vitro. Hematopoietic stem cells (HSCs), rare and quiescent cells that gives birth to all blood and immune cells in the bone marrow compartment, are under investigation for use in cell therapies and other regenerative medicine applications. Although fractions of cells can be enriched containing HSCs, all too often these subsets contain contaminating cell types that are not true stem cells. Lineage-restricted multipotent progenitors, for example, express the surface marker CD34, similar to HSCs, but do not exhibit the long-term multilineage graft potential that is indicative of a more primordial cell. Multiple markers have been analyzed at once in attempts to enrich solely for HSCs, but even these combinations, including CD38, Thy1, and CD45RA, are not adequate to do the trick. Researchers in John Dick’s lab have discovered a marker, CD45f, that seems to provide an even finer resolution while sorting for HSCs. Using flourescence-activated cell sorting, researchers enriched for a CD45f fraction and transplanted single cells into the femurs of mice. Many of these transplanted cells were capable of long-term multilineage engraftment. Sorting with CD45f allows for the highest purity of HSCs ever reported in the literature, coming in at around 9.5%.

Friday Science Review: July 15, 2011

New Target for AML

British Columbia Cancer Agency ♦ University of British Columbia ♦ Hannover Medical School ♦ Stanford University School of Medicine

Published in Cancer Cell, July 12, 2011

The MN1 locus is implicated in the development of acute myeloid leukemia (AML), where its up-regulation is a poor prognostic marker. Not all progenitors are transformed by MN1 over-expression though; common myeloid progenitors, rather than granulocyte-macrophage progenitors, are susceptible. In this study, researchers began to elucidate the genetic programs that underlie susceptibility to MN1 transformation. Complementation studies showed that MN1 required the MEIS1/AbdB-like HOX-protein complex to drive transformation. Chromatin immuno-precipitation illustrates that MN1 and MEIS1 have many identical chromatin targets, suggesting the two work together in some fashion to promote AML. Although MN1 relies on MEIS1 for transformation, it cannot activate expression of the complex, so transcriptional repression of MEIS1 could be an effective treatment paradigm for MN1-induced AML down the line.

De Novo Mutations in Non-Familial Schizophrenia

University of Montreal ♦ McGill University ♦ University of Hong Kong ♦ Others..

Published in Nature Genetics, July 10, 2011

In an eloquent study, researchers have identified a group of genes that could be responsible in part for the development of schizophrenia. The study involved 14 ‘trios’, including 14 schizophrenia patients and their parents. By focusing on non-familial cases (none of the patients had first- or second- degree family history of psychotic disorders), it could be assumed that the primary genetic events driving pathogenesis were the de novo mutations (DNMs)  occurring in the genomes of the patients themselves. The exomes of the 14 patients were sequenced which led to the identification of 15 DNMs in 8 of the patients, a much greater rate than previously reported. These findings suggest DNMs may contribute to the heritability of the disease and provide a list of targets for future consideration.

Mechanism of Carvedilol Elucidated

University of Calgary ♦ Rush University ♦ University of Iowa ♦ Thomas Jefferson University ♦ UC San Diego ♦ Others..

Published in Nature Medicine, July 10, 2011

One of the most potent beta blockers for the treatment of heart failure is carvedilol, however until now its mechanism of action was largely unknown. Researchers show that the small molecule suppresses a process known as store overload-induced calcium release (SOICR). When patients have tachyarryhythmias leading to heart failure, SOICR is usually the cause, and carvedilol was the only beta blocker under investigation that could suppress this calcium release. Carvedilol exerts its effects by reducing the period of time that cardiac ryanodine receptors are open for, and hence the amount of calcium that is released, preventing arrhythmia. To further investigate carvedilol’s SOICR-suppressing capabilities, researchers constructed an analog with vastly reduced beta blocking capacity and showed that it prevented stress induced ventricular tachyarrhythmias in mice. Combining the analog with selective beta blockers, like metoprolol or bisoprolol, produced optimal effects.

Using DNA to Program Quantum Dot Self-Assembly

University of Toronto ♦ Published in Nature Nanotechnology, July 10, 2011

Shana Kelley’s lab has impressed once again with this paper that illustrates how DNA can be used to not only guide the self-assembly of quantum dots, but regulate a series of properties that are crucial to their function in areas such as optical detection, solar energy harvesting, and biological research. Ideally quantum dots exhibit high luminescence efficiency, spectral tunability, control over valency (how many quantum dots a single dot is connected with), and bonding control. In this study, researchers constructed nanocrystals from cadmium telluride and capped them with specific sequences of DNA. The strategy for developing these ideal dots involved varying the brightness of dots, size of dots, the number of DNA strands per dot, and the sequences of those DNA strands. Combining these elements allows for broader functionality and a myriad of different construction possibilities, including cross-shaped forms containing three different dots. Although DNA-functionalized quantum dots have been made before, this is the first time they have been constructed with control over valency and other properties. Quantum dots can be switched on and off as well; modulating pH can alter conformation and the transfer of energy between dots in higher-order complexes.

Friday Science Review: July 8, 2011

Reductive DNA Damage, A New Evil

University of Waterloo ♦ University Health Network ♦ University of Toronto ♦ Published in PNAS, July 5, 2011

The mechanism behind oxidative DNA damage is well known. It has long been thought that oxidative damage causes the majority of DNA damage in a cell, leading to malignant transformation and cancer. New findings suggest that reductive damage is just as bad, if not worse, for the well-being of a cell. Scientists were able to observe ultrafast-electron-transfer reactions between a rare species of electron, known as a prehydrated electron, and different electron scavengers. These measurements are extraordinarily fast, occurring on the femtosecond time scale. When comparing the reductive damage done by electron transfer reactions involving prehydrated electrons, to the oxidative damage done by OH radicals, researchers came upon a rather surprising finding: the yield of reductive DNA strand breaks was roughly twice that of oxidative strand breaks, as measured using gel electrophoresis.

Clinical Study Results: Glutamic Acid Decarboxylase Vaccine in Patients with Type 1 Diabetes

Hospital for Sick Children (and other sites) ♦ Published in Lancet, June 27, 2011

Results from a clinical study run at multiple test centres in both Canada and the USA, indicate that an antigen vaccination targeting glutamic acid decarboxylase (GAD) was unsuccessful in treating type 1 diabetes. Although GAD is a major target of autoimmune response, preclinical data acquired through animal studies failed to translate into an effective treatment in the clinic. The patient group under examination included those 3 – 45 years of age, diagnosed with type 1 diabetes within 100 days. Patients received one of three courses of treatment: (1) three injections of GAD mixed with an adjuvant, (2) two injections of GAD mixed with adjuvant, and one injection of adjuvant alone, or (3) three injections of adjuvant alone.

The primary outcome of the study was the mean area under the curve of serum C-peptide during the first two hours of a meal tolerance test at one year. Patients in the group receiving three doses of the vaccine had a C-peptide value of 0.412 nmol/L at the one year mark, while those receiving three doses of the adjuvant alone had a value of 0.413 nmol/L; the loss of insulin secretion virtually unchanged.

Utilizing the host immune system to attack disease endogenously is a powerful paradigm upon which to build future therapies, but as is so often the case, finding relevant and predictive animal models is a serious limitation in their pursuit.

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.


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