Human enamel development of the permanent teeth takes place during childhood and stresses encountered during this period can have lasting effects on the appearance and structural integrity of the enamel. One of the most common examples of this is the development of dental fluorosis after childhood exposure to excess fluoride, an elemental agent used to increase enamel hardness and prevent dental caries. Currently the molecular mechanism responsible for dental fluorosis remains unknown; however, recent work suggests dental fluorosis may be the result of activated stress response pathways in ameloblasts during the development of permanent teeth. Using fluorosis as an example, the role of stress response pathways during enamel maturation is discussed. Dental enamel is produced by specialized epithelial-derived cells known as ameloblasts, which are a layer of tall columnar polarized cells, and is the product of ameloblast progression through the various stages of their life cycle. In order to produce enamel, ameloblasts must synthesize and secrete large amounts of different proteins. This makes enamel especially susceptible to perturbations in ameloblast protein synthesis, both from internal and external sources. Perturbations in protein synthesis and secretion from trauma or illness can be observed visually in the teeth of affected individuals as unusually prominent Striae of Retzius, which are visible manifestations of the ameloblast growth cycle analogous to the growth rings of a tree . Both feFluoride is used to increase enamel hardness and prevent the formation of caries by incorporation into the hydroxyapatite (HA) crystals that make up the bulk of dental enamel. Fluoride is not produced by the body and so must be provided as a supplement, either from community water programs or in the form of gels, coatings, or rinses supplied by dental professionals. The dose required for beneficial effects of fluoride is quite low with community water guidelines stating that 0.7 parts per million (ppm) is sufficient . At doseIn order to better understand the role of protein synthesis during amelogenesis, a brief review of ameloblast function and enamel development follows. Amelogenesis, or enamel development, occurs over five stages but two stages are arguably the most important: the secretory stage and the maturation stage (reviewed in ). In theIn the secretory stage, the enamel reaches full thickness but is composed of a soft, cheese-like substance easily separated from the dentin by mechanical techniques. This is due to the high levels of protein still found in the enamel matrix (reviewed in ). Due toBy the beginning of the maturation stage, the enamel has reached full thickness  and, in RE ameloblasts are readily identified by large invaginations on their apical surface, which are believed to simultaneously secrete the enamel protease KLK4, absorb degraded enamel matrix for intracellular breakdown ,23,24,25Amelx which result in X-linked amelogenesis imperfecta (AI) [Ambn−/− mice have a severe phenotype presenting severely malformed, very thin enamel [−/−Enam mice lack true enamel [To illustrate the importance of protein secretion during the development of healthy enamel, defects associated with secretion or production of any of these important proteins results in malformed enamel in both mice and humans (reviewed in ). For excta (AI) ,30. AI in enamel . No AI-cn enamel . Enam−/−e enamel  and in he enamel , in whice enamel . Afflicte enamel  or cariee enamel .Klk4-knockout produces enamel of normal thickness showing the decussating rod pattern; however, this enamel is quite brittle, breaking close to the dentino-enamel junction (DEJ) [Mutations affecting the enamel matrix proteases, MMP20 and KLK4, can also produce the AI enamel phenotype in an autosomal recessive manner ,37,38. Aon (DEJ) ,41,42,43Cftr), in addition to causing Cystic Fibrosis, produces thin malformed enamel in both mice and humans [In addition to the canonical enamel proteins, defects in members of other cellular processes can also produce a dysplastic enamel phenotype. AI has also been linked to mutations in two members of the family with sequence similarity (FAM) gene family, FAM83H and FAM20A, as well as WD repeat containing domain 72 (WDR72) ,45,46. Dd humans ,48,49, ud humans . Though Fluoride is a widely used supplement that increases enamel hardness and reduces the incidence of dental caries . In contFluorosed enamel contains more protein than normal enamel leading researchers to speculate that decreased enamel protease function in the presence of fluoride was to blame . HoweverFluoride increases oxidative stress by reducing the activity of antioxidant enzymes ,59,60 reNrf2−/− mice, the enamel from null mice was more sensitive to acid, leaching greater amounts of calcium than control teeth [Fluoride also induces expression of genes associated with oxidative stress . In osteol teeth . This cool teeth , which gol teeth . Fluorosol teeth , suggestAs previously mentioned, oxidative stress can decrease protein synthesis through the HSP90-eIF2 mechanism but high levels of ROS can also affect proteins in other ways. One way oxidative stress can also affect protein function is through a direct or indirect post-translational modification of amino acid side chains known as carbonylation. Carbonylation can both positively and negatively affect protein function (reviewed in ), dependThe unfolded protein response (UPR) has evolved to sense the accumulation of misfolded proteins and halt protein synthesis until the misfolded proteins are refolded or degraded (reviewed in Hetz ). UPR isFluoride has been shown to activate PERK in osteoblasts  and causTreatment of ameloblast lineage cells with fluoride induces eIF2α phosphorylation in vitro, which is increased under mildly acidic conditions, but the kinase responsible is currently unknown ,82. In aPhosphorylation of eIF2α does not halt all protein synthesis, mRNA species possessing alternate upstream open reading frames (uORFs) may still be translated . PhosphoAs mentioned above, normal enamel development includes oscillations in pH during the maturation stage, cycling between neutral (pH 7.4) and mildly acidic (pH < 6.0) , which rIn cultured ameloblast-derived cells treated with fluoride, proteins associated with cellular stress (JNK and c-jun) were more readily phosphorylated under acidic conditions compared to neutral conditions . These cThe importance of producing correctly folded and functional enamel proteins is demonstrated by the wide variety and abundance of enamel defects that are traceable back to a single aberrant protein (AI) or to disrupted protein synthesis . Likewise, understanding how particular compounds affect proper ameloblast function is equally important for two reasons: Firstly, for providing additional insight into the mechanism of enamel development, and secondly for developing methods to avoid the development of, or for the treatment of, existing enamel formation disorders.


Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and age-related macular degeneration (AMD), share two characteristics in common: (1) a disease prevalence that increases markedly with advancing age, and (2) neuroinflammatory changes in which microglia, the primary resident immune cell of the CNS, feature prominently. These characteristics have led to the hypothesis that pathogenic mechanisms underlying age-related neurodegenerative disease involve aging changes in microglia. If correct, targeting features of microglial senescence may constitute a feasible therapeutic strategy. This review explores this hypothesis and its implications by considering the current knowledge on how microglia undergo change during aging and how the emergence of these aging phenotypes relate to significant alterations in microglial function. Evidence and theories on cellular mechanisms implicated in driving senescence in microglia are reviewed, as are “rejuvenative” measures and strategies that aim to reverse or ameliorate the aging microglial phenotype. Understanding and controlling microglial aging may represent an opportunity for elucidating disease mechanisms and for formulating novel therapies. As the prevalence of such diseases, which include Alzheimer's disease, Parkinson's disease, and age-related macular degeneration (AMD), demonstrates a strong association with increasing age, aging changes in microglia have been hypothesized to play a prominent role in disease pathogenesis. Over the last few years, new and exciting discoveries with respect to microglial physiology and function have provided key insights that provide a conceptual framework for thinking about the connection between microglial aging and neurodegenerative disease. First, we have learned from studies employing parabiosis to create chimerism in circulating bone-marrow derived precursors that microglia are long-lived cells in the healthy CNS, with residence times that extend across much of an animal's life-span, and are therefore susceptible to aging factors This review aims to summarize what is currently known about: (1) the nature of the aging microglial phenotype, (2) how microglial aging can impact the function of microglia in their everyday roles, as well as in their response to perturbations, (3) what mechanisms underlie the generation of the aging microglial phenotype, and (4) whether aging changes in microglia may be successfully reversed .How do microglia in the uninjured and healthy CNS change with aging? Microglia in the healthy young CNS have a typical ramified morphology and are distributed throughout the neural parenchyma in a “space-filling” manner, providing efficient spatial coverage of the entire CNS milieu. This orderly arrangement of ramified microglia reflects the presence of organizing factors whose identity, mechanism, and purpose are still rather mysterious. Interestingly, as the CNS ages, the fidelity of these regulatory factors appears to diminish as the numbers, distribution, and morphological features of microglia progressively changes with time, reflecting aging-sensitive alterations in microglia function.The total number and density of microglia have been noted to increase significantly with age in various CNS compartments, including the hippocampus  using R-[11C]PK11195 revealed increased ligand binding in several cortical and subcortical areas, indicating increased levels of basal microglial activation  are detected in aged microglia studied Given the above descriptions for how microglia change in their structural and basal activation state with aging, how can we connect these aging phenotypes to potential changes in “everyday” functions of microglia? Concepts concerning the constitutive functions of microglia in a healthy CNS have been changing and developing in an exciting way over the last few years as key discoveries reveal new functional roles. A number of metaphors have arisen in the literature concerning these roles: microglia, for their role in dynamic immune surveillance, have been likened to “cops on the beat” Raivich, . For theAs the “constant gardener” of synapses, microglia in recent studies have been found to constitutively regulate synaptic structure and neuronal activity. The constant dynamic behavior of microglial processes  are not fully understood. Microglia can influence NPCs via secreted factors that direct their migration and differentiation -induced systemic infection in aged mice, inhibition of IL-1β signaling blocked both the neuroinflammatory response as well as the associated cognitive deficits  such as TLR1, TLR2, TLR4, TLR5, TLR7, and CD14 are up-regulated with increasing age  in the CNS. Mitochondria-derived ROS are produced in close proximity to mitochondria DNA (mtDNA) which encode components of the mitochondria electron transfer complexes. In aged microglia, mtDNA damage accumulates and leads to dysfunction in the respiratory chain and an over-production of ROS  for the treatment of neurodegenerative disease  that drive the emergence of senescent phenotypes suggest the feasibility of strategies that aim to ameliorate or reverse these changes. Measures targeted at lipofuscin accumulation in microglia may help alleviate lysosomal dysfunction, increase autophagy, decrease ROS production, and in general, ameliorate the detrimental phenotypes of aging microglia  has been found to promote mtDNA transcription and maintain its architectural structure   possible sources of replacement cells (including stem cells sources) (Selvaraj et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Natural Killer cells are a subset of cytotoxic lymphocytes that are important in host defense against infections and transformed cells. They exert this function through recognition of target cells by cell surface receptors, which triggers a signaling program that results in a re-orientation of the microtubule organizing center and secretory lysosomes toward the target cell. Upon movement of secretory lysosomes to the plasma membrane and subsequent fusion, toxic proteins are released by secretory lysosomes in the immunological synapse which then enter and kill the target cell. In this minireview we highlight recent progress in our knowledge of late steps in this specialized secretion pathway and address important open questions. Natural Killer (NK) cells and cytotoxic T lymphocytes (CTL) are essential effectors of innate and adaptive immunity against infected or nascent cancer cells . The cytThe lytic granule pathway involves the vectorial and polarized trafficking of the granules toward the immunological synapse and the re-organization of the microtubule organizing center toward the target cell and subsequent release of cytotoxic proteins. Besides the cytotoxic proteins, the granules also contain lysosomal enzymes, an acidic proteoglycan core, and lysosome-associated membrane proteins. Since this content is released by outside in signaling, lytic granules represent a hybrid organelle with shared properties of lysosomes and secretory granules and are therefore also called secretory lysosomes or lysosome related organelles , 5.RAB27A , UNC13D (FHL3), STX11 (FHL4), and STX-BP2 (FHL5), which encode proteins of conserved families that regulate membrane trafficking , whose cytotoxic lymphocytes fail to kill target cells. As a consequence of which patients suffer from uncontrolled and massive lymphocyte and macrophage proliferation. A group of genes has been discovered in which mutations causes strongly impaired lytic capacity. These include STX11 FHL, and STXEven though NK cells and CTL functions belong to distinct arms of immunity, mutations in genes causing FHL produce dramatic yet remarkably related cytoarchitectural and immunodeficiency phenotypes. In addition, primary CTL and NK cell responses reveal similar molecular requirements , 7. CollCareful electronmicroscopy studies in CTL, mast cells, and melanocytes revealed a heterogenous appearance of secretory lysosomes in terms of cytoplasmic localization, size, and electron dense luminal matrix –10. KineA second step in secretory lysosome maturation occurs when the cytotoxic lymphocyte engages itself in an interaction with a target cell during which the exocytic endosomes and associated exocytic machinery move to the immunological synapse in parallel with organelles containing LAMPs, perforin, and granzymes. At least two scenarios are compatible with the available data for the next stage in the secretory lysosome maturation pathway. The two organelles may fuse with each other to generate a structure with lytic and exocytic potential at the immunological synapse. Alternatively, tethering of the exocytic endosome equipped with munc13-4, rab27, and rab11, to the lytic precursor organelle with perforin and granzyme  is already sufficient to assemble and concentrate the protein machinery for efficient docking and fusion at the plasma membrane Figure . In suppMolecular insight into the question as to how munc13-4 could serve as a tethering molecule derived from computational structure predictions which revealed very weak homology between the MUN domain and subunits of intracellular membrane tethering complexes . The MUN2+- dependent PIP2 binding and the C2C domain might constitutively bind membranes, munc13-4 could bridge two membranes via this multiplicity of interactions. CTL and NK cells express two other rab27a effectors, slp1 and slp2-a. Both are non-essential for secretory lysosome release, but they could assist in tethering since their C2 domains are important for membrane binding and they focus tightly at the immunological synapse  on the partnering inner leaflet of the plasma membrane have not yet been defined. Given the analogies between cytokinesis, polarized exocytosis, and degranulation  we specuTethering of munc13-4, rab27, and rab11 structures with LAMP containing organelles can be considered as a pre-requisite for late endosome-lysosome fusion. In CTL and macrophages this heterotypic fusion event has been shown to involve the Qb SNARE vti1b, which is required for release of secretory lysosome content , 36, 37 In hemopoietic cells, syntaxin-11 interacts munc18-2 , 46, a min vitro reconstitution assays. Rizo’s lab recently developed an in vitro liposome fusion assay in which they could show for the first time a dependency of fusion on munc18 and munc13  and GTPase activating proteins (GAPs) for rab27a, and thereby the activity of the small GTPase are controlled upon signaling. This information could contribute insight in the order with which different rab27a effectors are recruited within a cell. We also need to improve our understanding where the proteins that control membrane traffic during degranulation are localized. Progress on this question will come from the application of powerful new imaging technologies that are increasingly being employed in immunological research. Ultimately such information will be crucial to annotate which step is controlled by which protein and how the network of traffic regulators cooperates in degranulation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe2+ as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well-documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms (1) indirectly via small RNAs, (2) binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and (3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.In the ancient anaerobic environment, ferrous iron (Fe Transition metals are essential elements in biological systems. Metabolic pathways, DNA synthesis, RNA synthesis, and protein synthesis are dependent on the availability of the appropriate metal cofactor. In support of this, all cells have designated gene products that transport metals to maintain cellular function; however, certain essential metals cause the formation of toxic reactive oxygen species (ROS). In the earliest description of what is now known as the Fenton reaction, iron (Fe) was shown to act catalytically in the oxidation of tartaric acid Fenton, . The Fen3+. Most siderophores are produced by the non-ribosomal peptide synthesis (NRPS) pathway and an example is the siderophore enterochelin. The final steps of the pathway are executed by the action of the Ent proteins  . Aerobactin is an example of a siderophore not produced by the NRPS pathway. Transcriptional control of both siderophores is regulated by the concentration of intracellular Fe2+  that constitutively expresses iron uptake proteins supported this hypothesis   and has been used to study Fur regulation for nearly 20 years  Figure  (De Lore) Figure . As a tr Hantke, . Fur rep Hantke, , 1987. I2+-dependent superoxide dismutase (SodB)  named ryhB in activation by Fur   and the Fe Hantke, , 2002. Ts Figure . DeletioS. Typhimurium, transcription of the virulence factor hilA requires Fe2+ through Fur-dependent regulation  assay we determined that Fur bound the upstream regulatory region of hns in a metal-dependent manner , and nuoA] contain Fur boxes located ≈100 bp upstream of the transcriptional start site, while the Fur-repressed tbp contains a Fur box that overlaps with the RNAP-binding site. The Fur box and activation of norB, which encodes a protein responsible for protection against NO  of RNAP, which promotes transcription of the target gene  and increases the intracellular Fe2+ concentration resulting in enhanced Fur activation  fur is not required for ftnA expression in the absence of hns; and (2) ftnA expression is not reduced by Fe2+-chelation in Δhns  by superoxide dismutases (SODs) and detoxification of H2O2 by hydroperoxidases . Unlike most pathogenic bacteria, S. Typhimurium contains 6 genes whose gene products are devoted toward degradation of H2O2. HPI (encoded by katG), HPII (encoded by katE), a Mn-dependent catalase (encoded by katN), an NADH-dependent alkyl peroxidase system (encoded by ahpCF), and two thiol specific peroxidases (encoded by tsaA and tpx). HPII and KatN are under positive regulation by the alternative σ factor RpoS, whereas HPI is induced by the redox sensing regulator OxyR during hydrogen peroxide stress  and Mn2+ for MnSOD (SodA) whereas heme is required for HPI and HPII function  and indirectly activates expression of the gene encoding the FeSOD   during bacterial infection. Innate cell activation by PAMPs initiates the synthesis of large amounts of Fe2+ sequestering proteins to limit the available Fe pool for the pathogen, known as “nutritional immunity”  and several research groups determined the contribution of the SLC11A1 locus to severity of infection within animal models , Fe2+, or cobalt (Co) and is important for acidification of the phagosome , which may contribute to the inability of fur mutants to cause disease within animal hosts.The Fur protein contributes to virulence in animal models for numerous bacterial pathogens Table . AlthougB. subtilis within the lab of John Helmann  uptake regulator (Zur) is a Fur family regulator that responds to Zn2+ and was discovered by two groups working with E. coli and Bacillus subtilis (B. subtilis) . This regulator, originally isolated from Rhizobium leguminosarum (Diaz-Mireles et al., 2+ as a corepressor. In contrast to Fur and Zur, the role of Mur in bacterial pathogenesis is less understood. However, genes regulated by Mur are important for virulence in the pathogen Brucella abortus (Anderson et al., Zinc (Zn2+, Zn2+, and Mn2+. Because of the anemia of inflammation and nutritional immunity exerted by the host during infection, metals are in low abundance in response to infection. In this metal-poor environment, the demetaleted Fur would allow for efficient acquisition of iron and enhances the fitness of the pathogen; however, deletion of fur most often results in partial or complete attenuation within animal models of infection. Notably, deletion of fur results in reduced expression of active enzymes responsible for defense against ROS, reduced expression of key metabolic pathways, and reduced expression of important virulence factors. This signifies that Fur's critical contribution to virulence may not be due to its classical role as a transcriptional repressor of metal acquisition (Figure The Fur family of transcriptional regulators control virulence, defense against ROS, and transport of Fen Figure , but to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has long been regarded that the primary function of fungal peroxisomes is limited to the β-oxidation of fatty acids, as mutants lacking peroxisomal function fail to grow in minimal medium containing fatty acids as the sole carbon source. However, studies in filamentous fungi have revealed that peroxisomes have diverse functional repertoires. This review describes the essential roles of peroxisomes in the growth and survival processes of filamentous fungi. One such survival mechanism involves the Woronin body, a Pezizomycotina-specific organelle that plugs the septal pore upon hyphal lysis to prevent excessive cytoplasmic loss. A number of reports have demonstrated that Woronin bodies are derived from peroxisomes. Specifically, the Woronin body protein Hex1 is targeted to peroxisomes by peroxisomal targeting sequence 1 (PTS1) and forms a self-assembled structure that buds from peroxisomes to form the Woronin body. Peroxisomal deficiency reduces the ability of filamentous fungi to prevent excessive cytoplasmic loss upon hyphal lysis, indicating that peroxisomes contribute to the survival of these multicellular organisms. Peroxisomes were also recently found to play a vital role in the biosynthesis of biotin, which is an essential cofactor for various carboxylation and decarboxylation reactions. In biotin-prototrophic fungi, peroxisome-deficient mutants exhibit growth defects when grown on glucose as a carbon source due to biotin auxotrophy. The biotin biosynthetic enzyme BioF (7-keto-8-aminopelargonic acid synthase) contains a PTS1 motif that is required for both peroxisomal targeting and biotin biosynthesis. In plants, the BioF protein contains a conserved PTS1 motif and is also localized in peroxisomes. These findings indicate that the involvement of peroxisomes in biotin biosynthesis is evolutionarily conserved between fungi and plants, and that peroxisomes play a key role in fungal growth. Peroxisomes are ubiquitous organelles in eukaryotic cells and typically contain enzymes involved in the β-oxidation of fatty acids and detoxification of reactive oxygen species. Additionally, peroxisomes are known to have various physiological functions based on their roles in diverse metabolic activities. For example, mammalian peroxisomes participate in the lipid biosynthesis such as ether phospholipids, and in the oxidation of amino acids and polyamines  toxin, and paxilline  grow via elongation of the hyphal tip to form straight primary hyphae with branches. The hyphae are divided into distinct cells by the formation of septa, and thus filamentous fungi are characterized by multicellularity. The septum is proposed to have several possible functions, including increasing the mechanical integrity of hyphae and division of mycelium into sections that undergo distinct developmental processes. However, septa do not completely separate adjacent cells in the hyphae due to the presence of a septal pore, which allows the passage of cytoplasm and organelles between adjacent cells Markham, . This inAspergillus oryzae grown on agar medium to hypotonic shock, which caused most of the hyphal tips to burst and lose the cytoplasmic constituents  significantly reduces the ability of this strain to prevent excessive cytoplasmic loss  at the C-terminus  synthase and 7, 8-diaminopelargonic acid (DAPA) synthase reactions (Muralla et al., Biotin is an essential cofactor involved in a number of carboxylation and decarboxylation reactions Knowles, . In eukaAspergillus species, mutants of the pex5 and pex7 genes are defective in protein import into the peroxisomal matrix due to the lack of PTS1 and PTS2 receptors, respectively (Hynes et al., Aspergillus species also exhibit growth defects when grown on glucose (Hynes et al., In A new model for biotin biosynthesis in fungi is proposed in Figure As described above, plant BioF protein functions as a KAPA synthase and was shown to be cytosolic by GFP fusion at the C-terminus (Pinon et al., Aspergillus peroxisome-deficient strains showing biotin auxotrophy exhibit abnormal polar growth (Tanabe et al., Plant biotin-auxotrophic mutants exhibit embryo lethality, indicating that biotin biosynthesis is vital for plant growth and development (Schneider et al., The primary function of fungal peroxisomes was long thought to be limited to the β-oxidation of fatty acids. During the two past decades, an increasing number of studies have unmasked the functional diversity of fungal peroxisomes (Pieuchot and Jedd, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MYCN is a proto-oncogene implicated to be directly involved in neuroblastoma development. Amplification of MYCN is associated with rapid tumor progression and poor prognosis. Novel therapeutic strategies which can improve the survival rates whilst reducing the toxicity in these patients are therefore required. Here we discuss genes regulated by MYCN in neuroblastoma, with particular reference to p53, SKP2, and DKK3 and strategies that may be employed to target them.Neuroblastoma is the most common extra-cranial solid tumor of childhood. Despite significant advances, it currently still remains one of the most difficult childhood cancers to cure, with less than 40% of patients with high-risk disease being long-term survivors.  MYCN amplification which occurs in ~25% of neuroblastoma, associating with rapid tumor progression and a poor prognosis  transcription factors which also includes c-MYC, and MYCL. Deregulated expression of Myc family members have been implicated in the genesis of several human cancers. Consistent with this, studies have shown that MYC contributes to numerous aspects of tumorigenesis including unrestricted cellular growth and proliferation, angiogenesis, inhibition of differentiation, metastasis, genomic instability, and reduced cell adhesion  within target gene promoters and the subsequent recruitment of multiple transcriptional coactivators. Heterodimerization with MAX is required for direct binding of MYC proteins to DNA. INK4Bp15 , reporter gene assays, and/or more recently Chromatin Immunoprecipitation (ChIP) analysis, a technique which allows specific protein-DNA interactions to be isolated. Indirect target genes of MYCN are genes which are altered as a consequence of other genes or pathways that are directly regulated by MYCN , a central sequence-specific DNA binding domain (DBD), a tetramerization domain, and a highly basic C-terminal regulatory domain. Nuclear export signals (NES) are located within both the N- and C-termini, and three lysine-rich nuclear localization signals (NLS) are located within the C-terminal , separated by 0–13 bp, located within the promoters of downstream target genes  non-coding RNA molecules involved in post-transcriptional regulation of gene expression  TP53 Mutation Databasep53 knock-in mice develop more aggressive and metastatic tumors in comparison to p53 null mice, and that the different p53 mutants are associated with distinct tumor patterns  compared to p53 mutations (15%) was observed. In all three cases with MDM2 amplification and 8/12 cases with a ARFp14 alteration, the abnormality was present in both the diagnostic and relapsed specimen, whereas in 5/6 cases where a p53 mutation was detected it was present in the relapsed specimen alone. This therefore suggests that for MDM2 amplification and ARFp14 alterations their presence predisposes to relapse whereas p53 mutations are acquired at relapse  leading to high levels of MDM2 expression has been found in some tumors and is associated with a poor prognostic outcome, including neuroblastoma  in the Myc family are known to play a paradoxical role in driving both cellular proliferation and inducing apoptosis, which is thought to provide a safeguard mechanism to prevent uncontrolled cellular proliferation and oncogenic transformation. This paradox is observed histologically by a high mitosis-karyorrhexis index, a combined index of both proliferation and apoptosis, in both human MYCN amplified neuroblastoma tumors  as previously reported for c-MYC  of these cell lines are MYCN amplified and predominantly established following previous cytotoxic therapy at relapse  that can lead to higher expression of MDM2 and greater inhibition of p53, is associated with poor survival in neuroblastoma, in particular stage 4 patients with MYCN amplification  mapping to 1p36.31, which is a region commonly deleted in neuroblastoma and frequently correlated with MYCN amplification , a serine/threonine kinase which plays a key role in mitosis and is expressed at high levels in a Figure . AURKA ha Figure , and proa Figure . There ia Figure . Interesa Figure .MYCN mRNA and regulating its stabilization and translation, thereby forming a positive feedback loop, critical for MYCN amplified neuroblastoma tumor cell growth and survival  locus which can lead to inactivation of the p53 pathway. In neuroblastoma, BMI-1 is shown to be essential for neuroblastoma tumorigenesis in vitro and in vivo, cooperating with MYCN via inhibition of MYCN driven apoptosis and downregulation of p53 expression  in neuroblastoma and which is associated with MYCN amplification and aggressive disease progression , spiro-oxindoles (MI compounds), benzodiazepinediones, isoindolinones, isoquinolinones, and thiophenes  is an F-Box protein and the variable substrate recognition component of the E3 ubiquitin ligase SCFCIP1  which enable SKP2 to directly bind to target substrates  mapping to −756 and −389 bp upstream of the transcriptional start site within the SKP2 promoter and drive transcription , has implicated the wide applicability of targeting SKP2 as a strategy to reactivate p53 and as pro-senescence therapy  is the first clinically approved proteasome inhibitor, and has been reported to induce p27KIP1 expression through degradation of SKP2  have been developed and used with success in preclinical and/or clinical studies thereby highlighting these inhibitors as a new class of cancer therapeutics  in 1/4 xenografts  is routinely used in the treatment of high-risk disease following high-dose chemotherapy and stem cell rescue, in a setting of minimal residual disease , and also DKK receptors Kremen 1 and 2 . The coin vitro transformation model of normal human fibroblasts, reduced DKK3 expression was subsequently observed in cell lines and tumors of several different cancer types including liver, lung, prostate, breast, osteosarcoma, and leukemia  have demonstrated that DKK3 induced apoptosis is mediated via ER stress triggered activation of JNK with subsequent reduction in anti-apoptotic BCL2, mitochondrial translocation of BAX, cytochrome C release, and activated caspase cleavage  which target the 3′UTR of DKK3 . The low expression of DKK3 in several human cancers combined with its cancer specific apoptosis inducing effects suggest that re-establishing DKK3 holds promise as a novel targeted cancer therapy. The use of demethylating agents to re-express miR-17-92 cluster to re-establish DKK3 expression in MYCN amplified neuroblastoma, or the use of agents which directly affect MYCN expression in neuroblastoma cells such as the BET inhibitor, JQ1 (Mertz et al., miR-122 is currently in phase II trials for the treatment of patients with Hepatitis C virus (Hu et al., Alternative strategies more specific to neuroblastoma may involve the direct targeting of the oncogenic ARF pathway in neuroblastoma has become increasingly evident and targeting this pathway through small molecule inhibitors of the MDM2-p53 interaction in neuroblastoma which remains predominantly wt even at relapse, is an attractive therapeutic option. Currently newer more potent MDM2-p53 antagonists are undergoing commercial development, which will be tested in suitable preclinical models of neuroblastoma leading to their evaluation in early phase clinical trials and hopefully eventually into standard neuroblastoma therapy.In recent years, the involvement of the p53/MDM2/p14The precise involvement of SKP2 and DKK3 in neuroblastoma will require further investigation and these targets are currently much less developed in terms of their therapeutic potential. Nevertheless their involvement in other cancers as well as neuroblastoma makes them a priority for commercial and academic drug development programs.MYCN amplified and non-amplified settings to fully evaluate their dependence on MYCN.It is envisaged that the therapeutic strategies which have been proposed in this review are most likely to be effective in combination with existing treatment regimens or other targeted agents and not alone. The increased understanding of neuroblastoma tumorigenesis and mechanisms of therapy resistance and relapse, advances in the development of murine models which more closely resemble high-risk metastatic and/or relapsed disease, and the drive towards targeted/personalized therapies, will hopefully provide increased opportunities to test out some of the paradigms mentioned in this review. When testing therapies directed against all these targets it will be important to do so both in This is an exciting time for neuroblastoma drug development and with many new targeted agents in the pipeline undergoing preclinical evaluation and early phase clinical trials it surely will not be long before one reaches frontline therapy and improves the outcome, whilst hopefully reducing long-term toxicity in patients with high-risk disease.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 1918 influenza pandemic was a major epidemiological event of the twentieth century resulting in at least twenty million deaths worldwide; however, despite its historical, epidemiological, and biological relevance, it remains poorly understood. Here we examine the relationship between annual pneumonia and influenza death rates in the pre-pandemic (1910–17) and pandemic (1918–20) periods and the scaling of mortality with latitude, longitude and population size, using data from 66 large cities of the United States. The mean pre-pandemic pneumonia death rates were highly associated with pneumonia death rates during the pandemic period . By contrast, there was a weak correlation between pre-pandemic and pandemic influenza mortality rates. Pneumonia mortality rates partially explained influenza mortality rates in 1918  but not during any other year. Pneumonia death counts followed a linear relationship with population size in all study years, suggesting that pneumonia death rates were homogeneous across the range of population sizes studied. By contrast, influenza death counts followed a power law relationship with a scaling exponent of ∼0.81  in 1918, suggesting that smaller cities experienced worst outcomes during the pandemic. A linear relationship was observed for all other years. Our study suggests that mortality associated with the 1918–20 influenza pandemic was in part predetermined by pre-pandemic pneumonia death rates in 66 large US cities, perhaps through the impact of the physical and social structure of each city. Smaller cities suffered a disproportionately high per capita influenza mortality burden than larger ones in 1918, while city size did not affect pneumonia mortality rates in the pre-pandemic and pandemic periods. The “Spanish” influenza A/H1N1 pandemic of 1918 was a major epidemic event of the twentieth century. The influenza A/H1N1 virus infected approximately a third of the world's population and resulted in over 20 million deaths worldwide Haemophilus Influenzae, which was identified in 1892, was the causative agent of influenza Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and H. Influenzae, to disease severity was poorly understood The role of influenza on seasonal increases in respiratory morbidity and mortality rates was not well understood when the 1918 pandemic struck, as the virus had not been identified. It was initially thought that Pfeiffer's bacillus, commonly known as It has been hypothesized that poor underlying health conditions, in part due to the occurrence of World War I, may have affected the severity of the 1918 pandemic. In particular, epidemiological studies have shown that geographical variation in baseline health conditions prior to the pandemic and the public health interventions implemented during the pandemic may partly explain the observed differences in pandemic mortality rates across cities, provinces, and countries We divided the 1910–1920 study period into a baseline pre-pandemic period (1910–17) and a pandemic period (1918–20), as several countries, including the US, experienced several waves of pandemic activity throughout 1918–1920 We calculated excess death counts associated with the pandemic as the death counts observed during pandemic years minus the average death counts for pre-pandemic years (1910–1917). We also calculated excess death rates per 100,000 inhabitants using our estimate of population size of each city for the corresponding pandemic year. We calculated Spearman non-parametric correlation coefficients between the mean pneumonia and influenza death rates during pre-pandemic and pandemic years across the 66 US cities.α, where D denotes number of deaths per year, N indicates city-level population size, and α is determined by the proportionality of population to mortality rate City size has been found to follow a simple scaling relationship with measures of energy consumption, economic activity, demographics, innovation, patterns of human behavior, infectious disease burden, among others A list of the 66 large US cities studied, latitude and longitude coordinates, and baseline demographic information are provided in We found a significant correlation between the mean baseline pneumonia death rate during 1910–1917 in the 66 US cities and the pneumonia death rate during each of the pandemic years from 1918 to 1920  . By contα of ∼0.81 , indicating that smaller cities systematically experienced higher death rates  whereas no significant trend was found in 1919 and 1920 (P>0.4). We found a moderate and significant correlation between population size and excess influenza death rates in 1918 across US cities , but this correlation was not significant in 1919 and 1920.For 1918, excess pneumonia death rates were not significantly correlated with the baseline pre-pandemic pneumonia death rates or population size. In 1919 and 1920, a number of cities experienced negative excess death rates.The main finding of this study is the existence of a correlation between baseline pre-pandemic pneumonia mortality rates in 66 US cities and influenza-specific mortality rates during the pandemic years of 1918–1920. It is interesting that cities of all sizes experienced high mortality rates in the pandemic period, provided they had high pre-pandemic mortality levels. Influenza and pneumonia mortality rates in each individual year between 1910 and 1917 were strongly correlated with mortality rates in 1918, indicating that this was not a spurious association.The statistical associations between influenza and pneumonia death rates may represent a strong biological interaction between influenza viruses and respiratory bacterial pathogens associated with pneumonia. A possible synergistic association between the influenza virus and bacterial pneumonia has been suspected for a long time in the context of the 1918 pandemic influenza In 1924 Tomanek and Wilson concluded that pneumonia mortality rates were stable in US cities from 1910 to 1920, except for 1918, using the same data as in our study. The authors commented that pneumonia is the terminal phase of a diverse group of diseases, resulting in large uncertainty in the diagnosis of “pneumonia” and “influenza” Our analysis of death rates and population sizes suggest that smaller cities suffered a disproportionately large mortality burden due to influenza in 1918, as compared with larger cities. These results could be explained by differences in health care, socioeconomic status, or baseline health status between cities. Our findings echo those of a study of the 1918 influenza pandemic in rural areas of England and Wales, where less populated areas experienced higher influenza death rates than more populous ones The role of baseline socio-demographic or geographical factors on the health impact of the 1918 influenza pandemic remains debated. Specifically, in a study of four British cities, no association was found between influenza attack rates and residential crowding There are some caveats associated with the nature of the data used in our study. First, differential mortality reporting and death coding practices, both spatial and temporally, could be potential sources of bias in our study. During the time period covered in this study mortality registration was functional in specific areas, mainly in large metropolitan areas, while registration in rural areas were in different developmental stages. The process of incorporation of vital statistics from states and cities was progressive and restricted to those areas with laws and ordinances requiring mandatory registration of deaths with a standard medical certificate. In the opinion of the Director of the Bureau of Census the data quality was satisfactory as early as in 1904 A better understanding of the biological and epidemiological processes that influence the interaction between influenza viruses and the bacteria associated with pneumonia  can provide the basis for better influenza treatment and prevention programs. This pathogen-interaction issue is not only a matter of historical interest. Today, the association between influenza and bacterial infection remains a cause of elevated morbidity and mortality during pandemic


The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues. Internal circadian clocks govern daily variations in gene expression, physiology and behavior. In mammals, the main circadian pacemaker resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. A complex interplay between cell-autonomous rhythmic properties of SCN neurons and their network organization ensures the robustness of this central clock Interestingly, clock genes also tick outside the SCN, both in the brain and peripheral organs Therefore, a key question is to understand how these multiple clocks get together within the organism, and how they adjust to daily changes of the external world. The ambient light-dark cycle and the feeding schedule are important time cues (zeitgebers) able to entrain circadian oscillators The pituitary gland is a tempting candidate to convey at least part of the SCN control to peripheral clocks. Indeed, the pulsatile secretion of pituitary hormonal products in the main bloodstream tightly depends on specific hypothalamic neurons that receive direct or indirect inputs from the SCN As reported previously Under long (16L:08D) and short (08L:16D) photoperiod, several changes were noted in the liver and pituitary gland, as compared to the 12L:12D condition. These changes mostly consisted in phase shifting, and reduced amplitude for some clock gene oscillations, without alteration in the overall waveforms at our time resolution. We thus decided to use cosinor analysis to evaluate the relative phase of transcript accumulation between each light condition. Globally, under the 16L:08D condition, a delay in the peak of expression could be observed for each clock gene in both organs . The calInterestingly, although we also observed phase-shifts, the response to short photoperiod differed between pituitary and liver oscillators. Indeed, under the 08L:16D schedule, the circadian clock genes reached their maximum of expression earlier after light onset than under the 12L:12D cycle . HoweverThe gradual phase shifting observed for the liver clock when mice were raised under all three light-dark schedules roughly corresponded to the increase of day length. This prompted us to verify whether the gene expression profiles obtained under 08L:16D and 16L:08D regimen could be superposed with that from the 12L:12D condition, simply by sliding them by 4 hours toward the right or left hand, respectively. Indeed, the re-plotting of our data with the time origin defined as light offset instead of light onset, revealed a clear alignment of gene patterns in the liver . To estiOn the contrary, the re-plotting of data from pituitary glands on a new timescale based on light offset did not improve the alignment of circadian clock genes under either photoperiod . GloballTo test this hypothesis that meal time would be a weak entraining cue for the pituitary circadian oscillators, we submitted mice to temporal feeding restriction under a 12L:12D cycle. In a preliminary experiment, normally nocturnal mice were fed during the light phase exclusively , over one week. Remarkably, we observed a dramatic reduction in the amplitude of circadian clock gene oscillations in the pituitary gland (data not shown). By contrast, the profiles in the liver were completely phase-inverted as previously reported In order to ascertain that the pituitary clock eventually reached a steady-state over the course of the restricted feeding protocol, we repeated this experiment and submitted mice to either DRF or nighttime restricted feeding  during three consecutive weeks. Not surprisingly, a complete inversion of clock gene expression profiles was noted between the liver of mice subjected to DRF and NRF (data not shown), as depicted by an average phase shift of 12.52±0.34 hrs . Strikingly, after three weeks of DRF, the gene patterns were altered but still not reversed in the pituitary gland. Instead, all transcripts accumulated under DRF at average levels similar to those measured under the NRF protocol, but exhibited a marked loss of overt rhythmicity . Hence, Because of the heterogeneous composition of the pituitary gland, which contains not less than five different endocrine cell types, the damped rhythms observed in the gland of mice subjected to DRF could result either from a composite response of cell oscillators that would be reset or not by meal time, or from an homogeneously weak entrainment of all pituitary clock cells by food. In order to address this issue, we investigated the pituitary response to DRF at the cellular resolution. Under NRF, the PER1 protein followed a clear rhythm of expression throughout the gland . PER1 waA pioneer study reporting the entrainment of peripheral clocks by meal time revealed that circulating glucocorticoids inhibited phase-shifting of the liver clockwork under DRF, as revealed by a faster inversion of clock gene expression patterns in the liver of adrenalectomized mice as compared to sham-operated animals In accordance with previous report Altogether, our results reveal substantial differences in the regulation of circadian clock gene expression between the liver and the pituitary gland. Earlier studies already reported that the photoperiod The use of cultured explants from transgenic rats expressing the Per1-luciferase reporter as a readout of the circadian clockwork suggested that lungs are not entrained by daytime restricted feeding, but instead remain phase-locked to the light cycle, similarly to the SCN In mammals, parabiosis experiments have elegantly established that the SCN regulate circadian oscillations in different peripheral organs via distinct pathways. Heart, spleen, and adrenal gland perceive SCN influence exclusively through neural messages, whereas other routes are involved in the entrainment of liver and kidney circadian oscillators A flexible relative phasing between different circadian oscillators must be a major corollary to the differential entrainment of multiple peripheral clocks by several time cues. The apparent synchronization of most peripheral oscillators, and their average delay of four to six hours as compared to the molecular rhythm in the SCN To some extent, the dual entrainment by light and food is reminiscent of the theoretical model in which two mutually coupled oscillators, tracking dusk and dawn, respectively, are supposed to encode photoperiod Thus, the pituitary gland emerges as a compendium of the complexity of peripheral circadian oscillators. The circadian clockwork, expressed in most pituitary cells, may pace a wide number of functions through the various hormones released by the gland. For example, one may speculate that the most appreciable rhythm in PER1 expression in corticotrophs, as compared to other pituitary cell types, might contribute to the circadian activity of the HPA axis. However, the rhythmic clock gene expression in the adrenals does not appear to depend on the pituitary gland All animal studies complied with the animal welfare guidelines of the European Community. They were approved by the Direction of Veterinary Services of Hérault, France (Authorizations #34-383 and C34-172-13).Adult male C57/Black6 mice were purchased from Charles River  and raised under the indicated photoperiod for at least two weeks. Food was given ad libitum, except during restricted feeding protocols, as specified. Mice had free access to tap water.Before adrenalectomy, analgesia was obtained by an intra-muscular injection of ketoprofen (5 mg/kg). Surgery was performed via a dorso-lumbar approach under isoflurane anaesthesia. The adrenal glands were identified, removed or left in situ (sham), and the incision was closed. Operated animals were allowed to recover during 2 weeks before the restricted feeding protocol, with free access to a 0.9% NaCl solution to preserve their osmotic balance in absence of a secreting source of mineralocorticoids.GAAAGAAACCTCTGGCTGTTCCT and GGAATGTTGCAGCTCTCCAAA; Per2, ATCAACCCGTGGAGCAGGAA and GGGAGCTGCGAACACATCCT; Cry1, GTTCGCCGGCTCTTCCA and ATCCTCAAGACACTGAAGCAAAAA; Cry2, GGGACTCTGTCTATTGGCATCTG and GTCACTCTAGCCCGCTTGGT; Bmal1, GCAGTGCCACTGACTACCAAGA and TCCTGGACATTGCATTGCAT; Clock, CACAGCGGAGGTCGTCCTT and GACATCGCTGGCTGTGTTAATG; Npas2, CACTCGGAAAATGGACAAAACC and TGAGACTTCATTGTGTTTCTGCAA; Gapdh, GGAGCGAGACCCCACTAACA and ACATACTCAGCACCGGCCTC.On the day of experiment, the animals were sacrificed every 4 hours by cervical dislocation. Sham-operated and adrenalectomized mice were sacrificed every 6 hours, and their trunk blood collected for subsequent corticosterone assay Mice received an overdose of pentobarbital and were perfused transcardially with 4% paraformaldehyde. Pituitary glands were dissected, and 50-µm thick coronal sections were prepared with a vibratome. Free-floating pituitary sections were incubated with anti-PER1 primary serum . The circadian phase and mean level of each gene profile were then estimated by cosinor analysis, using the following equation in the curve fitting: f(t) = M+A. cos (2π. (t - ϕ)/24), .Statistical analysis was performed with the Prism 5 software (GraphPad). The liver and pituitary circadian oscillators were compared by Wilcoxon matched-pairs signed rank test, where the in liver and pituitary expression profiles of each gene were matched pairs. Circulating corticosterone levels in sham-operated and adrenalectomized mice were compared with two-way ANOVA, where the operation protocol and the time of sample collection were the variables. All values in the text are expressed as mean ± standard deviation. In the figures, transcript accumulation data are plotted as mean ± standard error of the mean.Figure S1Expression of the circadian clock protein PER1 in the mouse pituitary gland. Accumulation of PER1 protein was assessed by immunofluorescence in pituitary sections from mice submitted to nighttime feeding (NRF) or daytime feeding (DRF), sacrificed eight  or twenty  hours after light onset. (A) Under NRF, PER1 (red) is barely detectable at ZT8, and expressed throughout the gland at ZT20. PER1 is expressed in ACTH-containing cells  and other cell types (arrowheads). (B) Under DRF, expression levels of PER1 were similar throughout the gland at both time points. Insets show magnified details from merged images.(TIF)Click here for additional data file.


Voltage gated potassium channels have been extensively studied in relation to cancer. In this review, we will focus on the role of two potassium channels, Ether à-go-go (Eag), Human ether à-go-go related gene (HERG), in cancer and their potential therapeutic utility in the treatment of cancer. Eag and HERG are expressed in cancers of various organs and have been implicated in cell cycle progression and proliferation of cancer cells. Inhibition of these channels has been shown to reduce proliferation both in vitro and vivo studies identifying potassium channel modulators as putative inhibitors of tumour progression. Eag channels in view of their restricted expression in normal tissue may emerge as novel tumour biomarkers. Cancer is one of the major killers throughout the world. It is estimated that a total of 1,529,560 new cancer cases and 569,490 deaths from cancer will occur in the United States in 2010 [+channels comprise the largest family of ion channels encoded by ~300 genes with phenotypic diversity generated through alternative splicing, variable association of (homo/heteromultimerisation) of channel subunits and posttranslational modifications. In normal cellular function, K+ channels are the main determinants of a cell's resting membrane potential. K+ channels have also been linked to cell volume control[Ion channels are integral membrane proteins that mediate the transfer of ions through the hydrophobic lipid bilayer of the cell membrane. They play an important role in a variety of functions that range from nerve/muscle excitation , regulate control,7, cell e control and carde control.+ channel subtypes has been described in a plethora of malignancies. In particular the role of voltage gated K+ channels in cancer, has been reviewed in several excellent publications [+ channels with their potential therapeutic applications in cancer.In recent years, expression of several Kications ,10,11. TEag gene, present on locus 50 of the X chromosome of the fruitfly Drosophila melanogaster, is a mutant of the Shaker gene [Eag genes in Drosophila and mammals, a further two- Elk (Eag like gene) and Erg (Eag related gene) were discovered. All members of the Eag family have >85% DNA sequence homology [The ker gene , so callker gene ,14. In ahomology . The Inthomology The Eag channel has also been cloned from rat (rEag) , and bovThe human HERG gene was the first member of the Ether-a go-go family to be isolated by screening of human hippocampal cDNA with the mouse homologue of Eag and was localised to chromosome 7 . It has Eag channels are expressed in fusing myoblasts and been posulated to have a role in their hyperpolarisation that preceeds their fusion . Eag chaDrosophila melanogaster larvae is well known [shal and shaker channels in the central nervous system of Drosophila melanogaster have been shown to be reciprocally regulated resulting in a target dependent, homeostatic modulation of synaptic transmission [Drosophila [The function of Eag channels in neurotransmitter release at the neuromuscular junctions to initiate action potential in ll known  and recesmission . Eag chasmission  and are osophila . In mammosophila .In contrast to Eag channels, HERG channels are more widely expressed and their functions differ according to their localization Table . The HERDrosophila, mouse, rat and humans. The sequence comparisons among family members has shown that two members of the same subfamily in different species share about 60-70% amino acid identities from S1 through to the cNBD segment [Members of the Eag family share the same structure of other voltage-gated potassium channels, comprising of four identical α subunits each consisting of six membrane spanning domains (S1-S6) with cytoplasmic amino (N) and carboxy (C) termini. The ion-conduction pathway or pore region (P) is positioned between S5-S6, with voltage being sensed predominantly by the chain of positive arginine or lysine residues based at every third position, separated by two hydrophobic residues within the S4 domain which acts as a voltage sensor ,32. All  segment .herg mRNA were present in 17 cancer cell lines of different species (human and murine) with distinct histogenesis. These included neuroblastoma, rhabdomyosarcoma, adenocarcinoma, lung microcytoma, pituitary tumours, insulinoma B cells and monoblastic leukaemia [The initial study reporting on a potential link between the Eag family of channels and cancer showed that high levels of eukaemia .herg mRNA is also expressed in MCF-7 (breast cancer), SHSY-5Y (neuroblastoma) and He-La (carcinoma cervix) cell lines. Inhibition by antisense oligonucleotides decreased the RNA content and functional protein of EF119 (breast cancer) cells [Following this discovery, Walter Stuhmer's group showed that Chinese Hamster ovary (CHO) cells when transfected with rEag exhibited a transformed cancerous phenotype characterised by the ability of the cells to grow in a low concentration (0.5%) of serum, displaying increased DNA synthesis, higher metabolic activity and loss of contact inhibition . The samr) cells . Furtherr) cells . Eag char) cells . Additior) cells . These eEag channels have also been shown to have increased expression in various cancer cell lines namely IGR1, IPC298, and IGR39 (melanoma) , SH-SY5YHERG channels have been shown to be expressed in various cell lines like human and murine neuroblastoma, human leukaemia  -51, humaherg mRNA and HERG protein was expressed in 67 and 82% of endometrial cancer tissues compared to 18% of normal endometrium with no expression seen in endometrial hyperplasia [herg gene and HERG protein were expressed in blast cells of acute myeloid leukaemia patients while no expression was seen in peripheral blood mononuclear cells [herg transcript and HERG channels have been suggested to a play a role in prolactin secretion [The first tissue expression of HERG channels in cancer showed that erplasia . The samar cells . Similarar cells . Prolactecretion .herg gene and HERG protein in colorectal cancers, highest expression is seen in metastatic cancers with absence in normal colon and adenomas. HERG channels also modulate the invasiveness of colon cancer thought to be directly related to the amount of HERG protein present on the cell membrane [herg gene by siRNA resulted in reduced proliferation and invasiveness of the cells. In contrast high grade gliomas have shown lower expression of herg gene compared to high grade tumours [Further investigation of HERG channels in cancer invasion and metastasis revealed that, in addition to the high expression of membrane  and confmembrane  and melamembrane . Increas tumours  while th tumours .These studies show that Eag and HERG channels are expressed by a variety of cancer cell lines and tissues with Eag channel showing an oncogenic potential while HERG channels are associated with more aggressive tumours and have a role in mediating invasion.Eag has been shown to have high expression in colorectal cancers compared to adenomas and its expression correlates with tumour size, lymph node metastasis and Dukes staging suggesting its role as a prognostic marker . SimilarN-methyl-N-nitrosourea (MNU) compared to chemically induced Dextran sulphate sodium (DSS) colitis. These carcinogens are well known to induce premalignant changes in the colon mucosa. Moreover there was higher Eag protein expression and mRNA in the distal mouse colon treated by DMH and MNU compared to the untreated proximal colon which suggests their role in pathogenesis of colon cancer [Eag channels have been found to be up regulated in mouse colon on treatment with chemical carcinogens such as Dimethylhydrazine (DMH) and n cancer . Estrogen cancer . The samn cancer . Other fn cancer  and Aracn cancer .The potential of using Eag channel expression as a tumour marker is supported by observation that Eag channels show higher expression in all patients with cervical cancer . In the Injection of poly-lysine containing recombinant anti Eag1 antibody conjugated to Cy5.5 into immune deficient mice grafted with MBA-MB-435 S mammary cancer cell line clearly showed the tumour and the sentinel lymph node on near infrared fluorescent imaging (NIF) in 24 hours. .The increased expression of Eag channel in the mouse colon as a result of DMH exposure has been shown to be associated with poor survival. Eag has also be shown to be present at premalignant stage in the development of colon cancer therefore Eag transcripts present in stool samples and rectal biopsies may be useful as diagnostic and prognostic markers . Thus EaThe next question now arises: what is the role of these channels in proliferation and cell cycle and how are they associated with carcinogenesis? We have now started to get some answers but still are quite far away from determining their exact role in carcinogenesisThe indication of an erg like inward rectifier being involved in cell cycle came initially from neuroblastoma cells that showed current characteristics resembling those of erg channels with a rapid reduction in the current when the cells were synchronised in G0/G1 phase or G1/S boundary of the cell cycle . This no+) and caesium [Xenopus oocytes are a useful model for the study of the cell cycle as they are indefinitely arrested in the G2 phase of the first meiotic cycle, until a hormonal stimulus, for example progesterone, induces progression of meiotic division. Rat Eag (rEag) channels expressed in Xenopus oocytes reduce their activity when their maturation is induced by progesterone and also by Mitosis promoting factor as the oocytes progress through the cell cycle, denoting that Eag channels are cell cycle sensitive . The par caesium  which ma caesium . Human E caesium .herg gene is not detectable in peripheral blood mononuclear cells (PBMNC) and circulating CD34+ cells, but then is rapidly expressed as soon as they enter S phase on upon treatment with cytokine/growth factor mixture, suggesting that HERG channels play a role in cell cycle regulation [herg1 RNA in human myeloid leukaemias. Both HERG1 and HERG1b proteins were demonstrated on the plasma membranes and can form heterotetramers. The expression of these isoforms was found to oscillate during cell cycle, with HERG1 protein upregulated in G1 phase and down regulated in S phase, while the N truncated HERG1b isoform upregulated in S phase [The expression of gulation . Subsequ S phase accountin S phase .The Eag and HERG channels have been shown to be inhibited in tissues of varying histology by Eag and HERG blockers which are reviewed in ,11,72. I by hypoxia, subsequently leads to the transcriptional activation of genes encoding erythropoietin, VEGF and glycolytic enzymes, all thought to be involved in various aspects of tumour initiation, growth and metastasis . HEK cel+ influx through the channel. Eag channels also act as a scaffold for and activate Calcium -Calmodulin activated kinase II (CaMKII), forming a complex which remains active even in the presence of low calcium [There has been increasing evidence linking mutant Eag channels that contain non conducting subunits lacking functional pore with cell proliferation. Hegle et al  demonstr calcium , leading calcium .Activated Nuclear localization sequence (NLS) located on the C terminus of Eag channels results in activation of Mitogen activated protein kinase (MAPK) signal transduction pathway that regulates cell morphology . SarcomaFrom the above studies it is clear that blocking Eag and HERG channels inhibits cell proliferation and therefore disease progression. These channels have been demonstrated in the cell membrane by functional studies and therefore are accessible targets for modulation by drugs. Moreover Eag channels have restricted expression in the central nervous system, placenta and in myoblasts just prior to fusion but are expressed in cancer cell lines of various origin and cancer tissues making them a potential marker and target for various drugs ,22,85,86+ channels and share 47% of the amino acid sequence [Both Eag and HERG belong to the same family of voltage gated Ksequence . Thus ansequence ,29. Ther1. Chemical blockers: Imipramine and astemizole have been shown to abolish Eag currents and inhibit the cell proliferation of tumour cells and are easily available in the market for use ,88. Howe+ channel currents in isolated cells and also inhibit the growth of cancer cells from various organs both in vitro and vivo. Hence evidence in favour of this antibody may potentially be used either alone or in association with current established treatment to reduce the dose and associated side effects of conventional chemotherapeutic drugs [2. Monoclonal antibodies: These act as highly specific molecules for a targeted blockade of the channels and minimise the side effects associated with action on homologous channels. These antibodies may also be potentially used as vehicles for therapeutic agents for a site specific action . A monocic drugs .3. Inhibition of cell growth using small interfering RNA (si RNA) technologies: This is a potential new approach to knock down gene expression and reduce the amount of protein that is produced. The activity of Eag has been shown to be silenced by the use of Eag specific siRNA which result in reduced protein expression and inhibition of cell proliferation in various cancer cell lines with minimal non-specific side effects . The chaherg gene expression by the use of shRNAs for HERG1 and the HERG-1b isoform, reduced growth rate, cell viability and inhibited colony formation of neuroblastoma cells restricting them to G0/G1 phase of cell cycle. There was also inhibition of tumour cells injected into nude mice on treatment with sh RNA. Thus this technology can be potentially used in silencing of herg gene and subsequently the reduction in growth of tumour, but its effect on the heart needs to be evaluated and the delivery of these molecules to target organs still poses a significant challenge [1. Short hairpin (sh) RNA technology: The knock down of hallenge .2. Use of HERG blockers including E-4031 and ergtoxin have still not been tested in vivo studies but do show a promising role in potential use with chemotherapeutic agents or in chemoresistant disease. However tight cardiac monitoring will be needed due to the development of drug induced Long QT syndrome.Both Eag and HERG channels have been shown to be present in cancers of differing origin and have a role in cell proliferation, progression and survival. There is abundant data on the effects of various blockers on the inhibition of cell growth and these channels may prove to be promising novel therapeutic targets for the treatment for cancer. They can be potentially be used in conjunction with chemotherapeutic agents or can be used in chemoresistant disease to improve survival. Eag due to its restricted expression shows a promising role as a potential tumour marker.The authors declare that they have no competing interests.VA wrote the manuscript, RK and HS conceptualised the project and helped in preparation of manuscript, RS and AB corrected the manuscript.


Kashin-Beck Disease (KBD) is an endemic, chronic and degenerative osteoarthropathy principally occurring in children. The characteristic pathological change of KBD is chondrocyte necrosis in hyaline articular cartilage. Proteoglycans are one of the major components in the extracellular matrix of articular cartilage, and disrupted proteoglycan metabolism and loss of proteoglycans in articular cartilage from KBD patients has been observed. In this mini-review, we discuss the close relationship between chondrocyte death including necrosis and loss of proteoglycan, and its potential mechanism during KBD onset and development, which may provide new clues for KBD research. Kashin-Beck Disease (KBD) is an endemic, chronic and degenerative osteoarthropathy affecting approximately 3 million people in China , 2. It pPGs are one of the major components in the extracellular matrix (ECM) of articular cartilage. They are remarkably complex macromolecules consisting of one or several types of polysaccharide glycosaminoglycan (GAG) side chains and a unique core protein. Depending upon the nature of their GAG chains, PGs can be categorised as heparan sulphated PGs (HSPGs), keratan sulphate PGs (KSPGs), chondroitin sulphate PGs (CSPGs) and dermatan sulphate PGs (DSPGs). Alternatively, PGs can also be named according to their unique core proteins, such as aggrecan .+ and K+, which increases the osmotic pressure in the tissue. This contributes to the large water content present in the ECM of articular cartilage and thereby provides the biomechanical properties to resist the compressive loading applied to joints. Therefore, the function of articular cartilage is dependent on a high aggrecan concentration being present in the tissue ECM [Aggrecan is one of the major PGs distributed in the ECM of articular cartilage. It contains both CS/DS and KS GAG side chains attached to an extended core protein. Several aggrecan monomers can bind to hyaluronan, and this non-covalent interaction is stabilised through an additional interaction with link protein so forming stable ternary complexes, especially in the pericellular and territorial matrix around chondrocytes in articular cartilage . Aggrecassue ECM . In degessue ECM  and is ci.e. PG loss and matrix destruction have been proposed and studied in KBD.Mechanisms of PG loss in OA and RA have been elucidated and involve increased presence of the inflammatory cytokines interleukin 1 (IL-1) and tumour necrosis factor (TNF) that induce chondrocytes and synovial cells to produce and secrete Matrix MetalloProteinases (MMP) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS) proteinases that are responsible for the degradation of collagens and PGs, respectively . The patEvidence for PG catabolism in KBD patients has been provided from several previous studies in which GAG depletion has been observed in the articular cartilage from KBD patients , 9, indiThe mechanism of PG depletion from articular cartilage of KBD patients is not clearly understood. However, studies suggest that increased catabolism of aggrecan by ADAMTS4 and/or TS5 in articular cartilage may be involved as an elevated expression of BC-13, a neo-epitope generated by aggrecanases through cleavage of the interglobular domain within the core protein of aggrecan, has been observed in articular cartilage from both juvenile and adult KBD patients . MMPs maMoreover, inflammatory cytokines IL-1 and TNF may be involved in PG catabolism in KBD patients as elevated levels of these cytokines have been reported in synovial fluid and serum from patients diagnosed with KBD , 14–17. Increased catabolism of PG’s in articular cartilage is postulated to cause an increase of GAG fragment release in body fluids such as urine. Analysis of GAG excretion and their molecular weight in urine has been used to ascertain them as potential biomarkers for OA and RA , 19. SimChondrocyte death including necrosis  and apopNecrosis is a form of traumatic cell death that results from external factors such as infection, toxins, or trauma. It is accompanied by the early loss of membrane integrity, swelling of cytoplasm, and release of different degradative enzymes including MMPs and ADAMTS proteinases into the adjacent tissue . TherefoApoptosis is a result of a programmed cell death, which is tightly regulated by cellular signalling events and leads to the orderly disintegration of individual cells with shrinking of cytoplasm, late loss of membrane integrity and no release of degradative enzymes . TherefoHowever, in the analysis of articular cartilage from adult KBD patients, apoptotic chondrocytes have been observed in the eroded areas where PG depletion is localised , indicatThe consequences of cell death in articular cartilage are not only those of PG depletion but also of altered tissue homeostasis. A decrease in chondrocyte number will compromise the tissues ability to repair, and this is an area requiring further research.The precise aetiology inducing chondrocyte cell death and altered PG metabolism in KBD patients is not clear, although several factors have been proposed including selenium deficiency , iodine In vitro studies using isolated chondrocytes from articular cartilage and exposure to mycotoxins including deoxynlvalenol (DON), T-2 toxin, nivalenol (NIV), hutenolide (BUT), alternariol methyl ether (AME) and monlliformin (MON) showed increased cell DNA damage, elevated cell mortality and altered PG metabolism. Similar findings have been reported in animal model studies of KBD [The first systematic study investigating the effects of different mycotoxins (found in KBD areas) on chondrocyte metabolism was performed by our labs . In vitrs of KBD –48. For s of KBD –47. In as of KBD .Interestingly, selenium deficiency alone cannot induce significant changes described above –47, and in vitro studies have shown some mycotoxin-induced changes in type II collagen at gene and protein levels [In contrast to PGs, mycotoxins did not significantly influence collagen metabolism in articular cartilage from these animal models, although several recent n levels –53. ThisThe fact we must emphasize here is that there is no direct evidence between cell death and PG depletion in the articular cartilage in these animal model studies –48, becain vivo study [The mechanism by which these two risk factors work together is not clear. Previous studies have shown that mycotoxins including T-2 toxin mainly target on cell membranes by increasing oxidative stress within cells , suggestvo study , and mayin vitro [in vitro [in vivo [Alternatively, mycotoxins could disrupt PG metabolism directly. It has been reported that T-2 toxin significantly inhibited aggrecan gene and protein expression in chondrocytes cultured in vitro , 57, butin vitro , 59 and [in vivo , similar[in vivo , 61, 62.[in vivo , 64, wheMycotoxins may also interfere with GAG metabolism and subsequently influence PG maintenance and function in articular cartilage. Recently, in our lab an immunohistochemical study using CS/DS motif antibodies has been carried out on articular cartilage taken from rats administered a low selenium diet and/or T2 toxin. This study indicates that there is altered expression of CS/DS GAG chains under the different experimental conditions Fig. , providiOne unique pathological change in the articular cartilage from KBD patients is the “focal” cell death and its associated “localised” loss of PGs in the ECM. Similar changes have also been observed in different KBD animal models , 65, 66 There is a distinct compressive loading (from 1 MPa to 25 MPa) applied to the articular cartilage in major joints such as the knee , which wCurrently, there are no specific treatment methods for KBD patients available due to the unknown aetiologies of this disease. However, based on the selenium deficiency and mycotoxin contamination hypotheses, a series of measures have been performed in KBD endemic areas including provision of the susceptible population  with dietary grain imported from non-KBD areas and selenium supplements , which hAlthough KBD has been known and studied for many years, the precise pathological mechanism of the disease is still poorly understood. It is now believed that KBD is not caused by a single risk factor but by multi-risk factors. The disease is characterised by pathological changes in the articular cartilage by focal chondrocyte death (necrosis) and an associated PG depletion. This may be derived from the risk factor-induced disruption of PG metabolism and subsequent abnormal biomechanical strain distribution applied on chondrocytes. In this mini-review, we propose mechanisms to explain how chronic matrix remodelling processes and cell death contribute to structural damage that characterizes KBD. More research is required to fully characterize the detail mechanism of KBD.


Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release.NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse.These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells. Cells of the immune system defend the body against pathogens. The immune response can be broadly divided into the innate and adaptive responses, with cells of the innate system providing an initial broad-specificity response to pathogens and cells of the adaptive system providing a later, more specific response. Differing recognition patterns of innate versus adaptive cells is accomplished via the use of distinct receptors. Cytotoxic T lymphocytes (CTLs), natural killer (NK) cells and invariant NKT (iNKT) cells are cytolytic cells of the immune system that are able to destroy cells infected with subsets of intracellular pathogens. NK and iNKT cell receptors provide broad recognition for the innate response via a limited repertoire of activating and inhibitory NK cell receptors (for NK cells)  or a semPrecise delivery of the lethal hit is vital to ensuring that only the recognised target is destroyed while innocent bystander cells are left unharmed. CTLs use an unusual mechanism to direct secretion to the point where the T cell recognises its target . CTLs idRelease of cytolytic proteins is confined to a secretory cleft that forms in an area of otherwise tight membrane contact and provides a confined space in which perforin and other lytic proteins can be kept concentrated for attack on the target . The clePrevious studies of CTLs have revealed that not only does the centrosome direct polarisation of the secretory lysosomes to the immunological synapse, but the Golgi complex ,13-15 anWhile the role of the centrosome in directing polarised secretion has been well documented in CTLs, little is known about whether the centrosome contacts the plasma membrane in other cytolytic cell types of the immune system with perforin-containing secretory lysosomes. Both NK and iNKT cells polarise their secretory lysosomes towards the immunological synapses formed with antigen-presenting cells and can kill target cells, and the strength of signalling can modulate the degree of granule polarisation and killing of agonist-pulsed targets for iNKT cells . In thisThe docking of centrioles with the plasma membrane is most clearly visualised by imaging of contact sites using high-resolution EM. To investigate whether the same tight polarisation of centrosomes to the plasma membrane seen in CTLs occurs in other cytolytic cells of the immune system during target cell destruction, we therefore used high-resolution EM to examine the contact sites of NK cells conjugated to MHC I-deficient B cells and iNKT cells conjugated to lipid-pulsed, CD1d-bearing target cells .Since the secretory lysosomes of CTLs and NK and iNKT cells are the end points of the endocytic pathway, they were labelled by endocytosis of horseradish peroxidase (HRP), a fluid phase marker which accumulates in lysosomes and produces an electron-dense reaction product upon exposure to its substrate, 3,3'-diaminobenzidine (DAB). Continuous bulk HRP uptake also allowed earlier components of the endocytic pathway to be labelled in these cells, including early endosomes and recycling endosomes (which could be distinguished by their characteristic tubulovesicular morphology). NK and iNKT cells were labelled overnight with HRP to load the endocytic pathway, mixed with MHC I-deficient 722.221 target cells or lipid-pulsed human CD1d-expressing C1R target cells (CD1d-C1R), respectively, and the mixed cell populations were incubated at 37C to form conjugates. Cells were fixed at different time points during target cell killing, typically between 5-60 minutes for NK and 30-70 minutes for iNKT, and prepared for EM. Thin (50 to 100 nm) sections were analysed for the position of the centrioles in NK or iNKT cells that had formed synapses with their targets.Figure We observed tight centriole polarisation at the immune synapse in 61 conjugates formed by NK cells and their targets, and in 88 conjugates formed by iNKT cells with targets. To investigate the frequency of centrosome polarisation at the immune synapse in more detail, we measured the distance of the centriole from the contact site in all conjugates in which the centrosome was polarised towards the target and in which at least one centriole was visible within the section plane, within a single population of cells. Since thin sections only capture part of the centriole, and since a centriole has a length of 450 nm, we considered any centriole profile within 450 nm of the cell surface to be tightly polarised, although the majority of the centrioles appeared to be much closer than this. Forty-two NK cells and fifty iNKT cells with polarised centrosomes were analysed. Both NK and iNKT cells showed similar frequencies of tight centriole polarisation at the immune synapse: 14% of NK cells and 12% of iNKT cells showed centrioles greater than 1,000 nm from the contact site, 10% of NK cells and 18% of iNKT cells had centrioles 500 to 1,000 nm from the contact site and 76% of NK cells and 70% of iNKT cells showed tight polarisation. Thus, although there was some variation in actual centriole distance, the majority of cells showed tight polarisation.Images of fixed conjugates capture the interaction of cytolytic cells with their targets at different points during their interaction, allowing different stages of killing to be analysed, although the thin 50 to 100 nm) sections required for high-resolution electron microscopic analysis limit the number of organelles that can be captured in a single plane. Figure 0 nm sectIn both NK and iNKT cells, both the Golgi complex and the endocytic recycling compartment, defined as HRP-positive structures with tubular morphologies  and cultured on irradiated feeder cells in the presence of 2 μg/mL phytohemagglutinin  and 100 U/mL recombinant human IL-2  to obtain proliferation of polyclonal NK cells. All blood samples from healthy donors were obtained with their informed consent. Epstein-Barr virus-transformed human B cells (721.221 cell line lacking MHC I) were grown and maintained in culture in RPMI 1640 medium containing 10% FCS, and were used as targets for NK cells.Human iNKT cells were expanded from healthy donors' peripheral blood lymphocytes (PBL) with autologous dendritic cells pulsed with α-galactosylceramide   as described previously . Two wee6 cells/mL in growth medium lacking serum and used immediately for conjugation.To allow compartments of the endocytic pathway (including secretory lysosomes and recycling endosomes) to be identified in lytic cells during killing events, NK and iNKT cells were incubated for 14 to 16 hours immediately before conjugation, in 1 mg/mL HRP added directly to the growth medium as described previously ,36. C1R-en bloc staining in uranyl acetate as described previously [Lytic (NK or iNKT) cells were mixed 1:1 with appropriate targets, plated immediately into 24-well plates (0.5 mL/well) and incubated at 37°C for 5, 10, 20, 40 or 60 minutes (NK cells) or for 30 to 40 minutes or 60 to 70 minutes (iNKT cells) to allow cells to form lytic cell-target cell conjugates. Four separate conjugation experiments were performed for each cell type. iNKT cell samples were prepared in duplicate and three separate donors were used to provide NK cells. Similar results were observed for each experiment. Conjugates were fixed by the addition of 2× fixative  directly into the incubation medium and left for 10 minutes at room temperature. Fresh 1× fixative  was added and the samples were incubated for an additional 40 to 50 minutes at room temperature before being processed for DAB cytochemistry as described previously . NK cellFCS: foetal calf serum; IL: interleukin; PBS: phosphate-buffered saline.The authors declare that they have no competing interests.DP and MS generated and provided NK and iNKT cells, respectively. MS prepared iNKT and C1R-CD1d cells for conjugation. JCS performed all cell conjugation experiments, preparation of samples for electron microscopy and analysis by electron microscopy. JCS, GG and MS designed and discussed the experiments and results. JCS and GG wrote the manuscript. All authors read and approved the final manuscript.