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BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo,where activity of inputs can be differentially regulated,but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here,we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons,both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures,synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons,although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin,effective in treating type 2 diabetes and the insulin resistance syndromes,improves insulin resistance by reducing hepatic gluconeogenesis and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin use with decreased cancer incidence and cancer-related mortality. Furthermore,numerous preclinical and clinical studies have demonstrated anti-cancer effects of metformin,leading to an explosion of interest in evaluating this agent in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers associated with hyperinsulinaemia; however,metformin may also directly inhibit tumour growth. In this review,we describe the mechanism of action of metformin and summarise the epidemiological,clinical and preclinical evidence supporting a role for metformin in the treatment of cancer. In addition,the challenges associated with translating preclinical results into therapeutic benefit in the clinical setting will be discussed. View Publication

PURPOSE Bacillus anthracis infection (anthrax) can be highly lethal. Two recent outbreaks related to contaminated mail in the USA and heroin in the UK and Europe and its potential as a bioterrorist weapon have greatly increased concerns over anthrax in the developed world. METHODS This review summarizes the microbiology,pathogenesis,diagnosis,and management of anthrax. RESULTS AND CONCLUSIONS Anthrax,a gram-positive bacterium,has typically been associated with three forms of infection: cutaneous,gastrointestinal,and inhalational. However,the anthrax outbreak among injection drug users has emphasized the importance of what is now considered a fourth disease form (i.e.,injectional anthrax) that is characterized by severe soft tissue infection. While cutaneous anthrax is most common,its early stages are distinct and prompt appropriate treatment commonly produces a good outcome. However,early symptoms with the other three disease forms can be nonspecific and mistaken for less lethal conditions. As a result,patients with gastrointestinal,inhalational,or injectional anthrax may have advanced infection at presentation that can be highly lethal. Once anthrax is suspected,the diagnosis can usually be made with gram stain and culture from blood or tissue followed by confirmatory testing (e.g.,PCR). While antibiotics are the mainstay of anthrax treatment,use of adjunctive therapies such as anthrax toxin antagonists are a consideration. Prompt surgical therapy appears to be important for successful management of injectional anthrax. View Publication

Aldehyde dehydrogenases (ALDHs),enzymes responsible for detoxification and retinoic acid biosynthesis,are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date,however,there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay,and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor,diethylaminobenzaldehyde (DEAB),together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition,the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore,a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation,suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity. View Publication

Metformin,the first-line drug for treating diabetes,inhibits cellular transformation and selectively kills cancer stem cells in breast cancer cell lines. In a Src-inducible model of cellular transformation,metformin inhibits the earliest known step in the process,activation of the inflammatory transcription factor NF-κB. Metformin strongly delays cellular transformation in a manner similar to that occurring upon a weaker inflammatory stimulus. Conversely,inhibition of transformation does not occur if metformin is added after the initial inflammatory stimulus. The antitransformation effect of metformin can be bypassed by overexpression of Lin28B or IL1β,downstream targets of NF-κB. Metformin preferentially inhibits nuclear translocation of NF-κB and phosphorylation of STAT3 in cancer stem cells compared with non-stem cancer cells in the same population. The ability of metformin to block tumor growth and prolong remission in xenografts in combination with doxorubicin is associated with decreased function of the inflammatory feedback loop. Lastly,metformin-based combinatorial therapy is effective in xenografts involving inflammatory prostate and melanoma cell lines,whereas it is ineffective in noninflammatory cell lines from these lineages. Taken together,our observations suggest that metformin inhibits a signal transduction pathway that results in an inflammatory response. As metformin alters energy metabolism in diabetics,we speculate that metformin may block a metabolic stress response that stimulates the inflammatory pathway associated with a wide variety of cancers. View Publication

Proplatelet formation is a part of the intricate process by which platelets are generated by their precursor cell,the megakaryocyte. The processes that drive megakaryocyte maturation and platelet production are however still not well understood. The protein kinase C (PKC) family of serine/threonine kinases has been demonstrated as an important regulator of megakaryocyte maturation and proplatelet formation,but little investigation has been made on the individual isoforms. We have previously shown,in mouse models,that PKCα plays a vital role in regulating platelet function,so in this study we aimed to investigate the role of PKCα in megakaryocyte function using the same Prkca(-)(/)(-) mice. We assessed the role of global PKC and specifically PKCα in proplatelet formation in vitro,analyzed polyploidy in Prkca(-)(/)(-)-derived megakaryocytes and followed platelet recovery in platelet-depleted Prkca(-)(/)(-) mice. We show reduced proplatelet formation in the presence of global PKC blockade. However,in the presence of a selective classical PKC isoform inhibitor,Go6976,proplatelet formation was conversely enhanced. PKCα null megakaryocytes also showed enhanced proplatelet formation,as well as a shift to greater polyploidy. In vivo,platelet production was enhanced in response to experimentally induced immune thrombocytopenia. In conclusion,our data indicate that classical PKC isoforms,and more specifically PKCα,are negative regulators of proplatelet formation. PKCα appears to negatively regulate endomitosis,with the enhanced polyploidy observed in Prkca(-)(/)(-)-derived megakaryocytes. In vivo,these observations may culminate in the observed ability of Prkca(-)(/)(-) mice to recover more rapidly from a thrombocytopenic insult. View Publication

Bone remodeling,a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs),is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature,osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence,anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²?/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones,along with significantly more OBs and fewer multinuclear OCs. In vitro,although Camkk2?/? mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs,bone marrow cells from Camkk2?/? mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective,cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further,we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand,genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target,nuclear factor of activated T cells,cytoplasmic (NFATc1). Moreover,in vivo administration of STO-609 results in increased OBs and diminished OCs,conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall,our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis. View Publication

The future of safe cell-based therapy rests on overcoming teratoma/tumor formation,in particular when using human pluripotent stem cells (hPSCs),such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation,complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile,we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e.,survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors,leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular,a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g.,quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast,differentiated cell types (e.g.,dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together,these results provide the proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy." View Publication

INTRODUCTION Breast cancer stem cells are suspected to be responsible for tumour recurrence,metastasis formation as well as chemoresistance. Consequently,great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro,they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. METHODS We describe a sub-population expressing the stem-like marker CD44(+)/CD24(-/low) in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties,we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further,we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44(+)/CD24(low). Next,the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. RESULTS Using this method,Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore,the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. CONCLUSION In summary,we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A. View Publication

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication

Human cell transformation is a key step for oncogenic development,which involves multiple pathways; however,the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC),we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung,breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and,OCT4,in turn,stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover,we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a,promotes p63 but suppresses p53 expression,which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and,most likely,also to the iPSC process. View Publication