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New ultrabright fluorescent silica nanoparticles capable of the fast targeting of epithelial tumors in vivo are presented. The as-synthesized folate-functionalized ultrabright particles of 30-40 nm are 230 times brighter than quantum dots (QD450) and 50% brighter than the polymer dots with similar spectra (excitation 365 nm and emission 486 nm). To decrease non-specific targeting, particles are coated with polyethylene glycol (PEG). We demonstrate the in vivo targeting of xenographic human cervical epithelial tumors (HeLa cells) using zebrafish as a model system. The particles target tumors (and probably even individual HeLa cells) as small as 10-20 microns within 20-30 minutes after blood injection. To demonstrate the advantages of ultrabrightness, we repeated the experiments with similar but 200× less bright particles. Compared to those, ultrabright particles showed ∼3× faster tumor detection and ∼2× higher relative fluorescent contrast of tumors/cancer cells.

Despite the pivotal role of MYC in tumorigenesis, the mechanisms by which it promotes cancer aggressiveness remain incompletely understood. Here, we show that MYC transcriptionally upregulates the ubiquitin fusion degradation 1 (UFD1) gene in T-cell acute lymphoblastic leukemia (T-ALL). Allelic loss of ufd1 in zebrafish induces tumor cell apoptosis and impairs MYC-driven T-ALL progression but does not affect general health. As the E2 component of an endoplasmic reticulum (ER)-associated degradation (ERAD) complex, UFD1 facilitates the elimination of misfolded/unfolded proteins from the ER. We found that UFD1 inactivation in human T-ALL cells impairs ERAD, exacerbates ER stress, and induces apoptosis. Moreover, we show that UFD1 inactivation promotes the proapoptotic unfolded protein response (UPR) mediated by protein kinase RNA-like ER kinase (PERK). This effect is demonstrated by an upregulation of PERK and its downstream effector C/EBP homologous protein (CHOP), as well as a downregulation of BCL2 and BCLxL. Indeed, CHOP inactivation or BCL2 overexpression is sufficient to rescue tumor cell apoptosis induced by UFD1 knockdown. Together, our studies identify UFD1 as a critical regulator of the ER stress response and a novel contributor to MYC-mediated leukemia aggressiveness, with implications for targeted therapy in T-ALL and likely other MYC-driven cancers.

Cellulose acetate (CA), viscose, or artificial silk are biocompatible human-benign derivatives of cellulose, one of the most abundant biopolymers on earth. While various optical materials have been developed from CA, optical CA nanomaterials are nonexistent. Here we report on the assembly of a new family of extremely bright fluorescent CA nanoparticles (CA-dots), which are fully suitable for in vivo imaging/targeting applications. CA-dots can encapsulate a variety of molecular fluorophores. Using various commercially available fluorophores, we demonstrate that the fluorescence of CA-dots can be tuned within the entire UV–VIS-NIR spectrum. We also demonstrate excellent specific targeting of tumors in vivo, when injected in zebrafish (xenograft model of human cervical epithelial cancer), and unusually strong exvivo topical labeling of colon cancer in mice utilizing CA folate-functionalized nanoparticles., National Science Foundation (U.S.) (Grant CBET 1745530)

Characterization data of fluorescent nanoparticles made of cellulose acetate (CA-dots) are shown. The data in this article accompanies the research article "Ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications" [1]. The measurements and calculation of brightness of individual CA-dots are presented. The description of conjugation procedure Pluronic F127-Folic Acid copolymer and folic acid is shown. Identification of composition of CA dots using Raman and absorbance spectroscopy is demonstrated. The methods for image analysis of efficiency of CA-dot targeting of epithelial tumors xenografted in zebrafish is presented.

Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset in zebrafish without detectable effects on fish development. DLST is the E2 transferase of the α-ketoglutarate (α-KG) dehydrogenase complex (KGDHC), which converts α-KG to succinyl-CoA in the TCA cycle. RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of α-KG and a decrease of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Together, our studies uncovered an important role for DLST in MYC-mediated leukemogenesis and demonstrated the metabolic dependence of T-lymphoblasts on the TCA cycle, thus providing implications for targeted therapy.

Background Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by tumor heterogeneity, locoregional metastases, and resistance to existing treatments. Although a number of genomic and molecular alterations associated with HNSCC have been identified, they have had limited impact on the clinical management of this disease. To date, few targeted therapies are available for HNSCC, and only a small fraction of patients have benefited from these treatments. A frequent feature of HNSCC is the inappropriate activation of β-catenin that has been implicated in cell survival and in the maintenance and expansion of stem cell-like populations, thought to be the underlying cause of tumor recurrence and resistance to treatment. However, the therapeutic value of targeting β-catenin activity in HNSCC has not been explored. Methods We utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between β-catenin and cAMP-responsive element binding (CREB)-binding protein (CBP) using the small molecule inhibitor ICG-001. We generated and annotated in vitro treatment gene expression signatures of HNSCC cells, derived from human oral squamous cell carcinomas (OSCCs), using microarrays. We validated the anti-tumorigenic activity of ICG-001 in vivo using SCC-derived tumor xenografts in murine models, as well as embryonic zebrafish-based screens of sorted stem cell-like subpopulations. Additionally, ICG-001-inhibition signatures were overlaid with RNA-sequencing data from The Cancer Genome Atlas (TCGA) for human OSCCs to evaluate its association with tumor progression and prognosis. Results ICG-001 inhibited HNSCC cell proliferation and tumor growth in cellular and murine models, respectively, while promoting intercellular adhesion and loss of invasive phenotypes. Furthermore, ICG-001 preferentially targeted the ability of subpopulations of stem-like cells to establish metastatic tumors in zebrafish. Significantly, interrogation of the ICG-001 inhibition-associated gene expression signature in the TCGA OSCC human cohort indicated that the targeted β-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival. Conclusions Collectively, our results identify β-catenin/CBP interaction as a novel target for anti-HNSCC therapy and provide evidence that derivatives of ICG-001 with enhanced inhibitory activity may serve as an effective strategy to interfere with aggressive features of HNSCC. Electronic supplementary material The online version of this article (10.1186/s13073-018-0569-7) contains supplementary material, which is available to authorized users.

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later, more lethal stages of some cancers when chronically activated by endogenous ligands. However, other studies have suggested that, under some circumstances, the AHR can oppose tumor aggression. Resolving this apparent contradiction is critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA, shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors) approaches were used to confirm the hypothesis that AHR inhibition reduces human cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden chambers), migration (scratch wound assay) and metastasis (human cancer cell xenografts in zebrafish). Furthermore, these assays were used for a head-to-head comparison between AHR antagonists and agonists. AHR inhibition or knockdown/knockout consistently reduced human ER−/PR−/Her2− and inflammatory breast cancer cell invasion, migration, and metastasis. This was associated with a decrease in invasion-associated genes (e.g., Fibronectin, VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously associated with decreased tumor aggression. Paradoxically, AHR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3′-diindolylmethane) similarly inhibited irregular colony formation in Matrigel and blocked metastasis in vivo but accelerated migration. These data demonstrate the complexity of modulating AHR activity in cancer while suggesting that AHR inhibitors, and, under some circumstances, AHR agonists, may be useful as cancer therapeutics.

Extracellular vesicles (EVs), secreted by cells and found in body fluids play important roles in intercellular communication. Therefore, EVs are receiving increasing attention as potential biomarkers in the diagnosis and prognosis of various diseases. However, the detection and the quantification of EVs are hampered by the nanometer scale of these particles and by the lack of optimized quantification methods. Atomic force microscopy (AFM) is a powerful technology that can detect small particles. Here we report a 3D capture method for sample preparation of AFM which improves the accuracy, sensitivity and reproducibility for EVs’ detection, compared to conventional sample preparation methods. By shaking a mica plate in EV solution, all the EVs were captured onto the 2D surface. The majority of the captured particles have a size ranging from 10 to 120 nm, which correlates with size data obtained from transmission electron microscopy studies. This novel sample preparation method has high adaptability potential and can also be applied to other organic and inorganic nanoparticles.

Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

Genetic analysis of mutants deficient in the biosynthesis of the photosystem I complex has revealed several nucleus-encoded factors that act at different post-transcriptional steps of chloroplast gene expression. Here we have identified and characterized the gene affected in the tab 1-F15 mutant, which is specifically deficient in the translation of the photosystem I reaction center protein PsaB as the result of a single nucleotide deletion. This gene encodes Tab 1, a 1287 amino acid protein that contains 10 tandem 38-40 amino acid degenerate repeats of the PPPEW/OPR (octatricopeptide repeat) family, first described for the chloroplast translation factor Tbc2. These repeats are involved in the binding of Tab 1 to the 5'-untranslated region of the psaB mRNA based on gel mobility shift assays. Tab 1 is part of a large family of proteins in Chlamydomonas that are also found in several bacteria and protozoans, but are rare in land plants.

Membrane fusion depends on the formation of a complex of four SNARE motifs, three that bear a central glutamine and are localized in the target membrane (t-SNARE) and one that bears an arginine and is localized in the donor vesicle (v-SNARE). We have characterized the arginine 56 to proline mutant (R56P) of synaptobrevin-2 (Sb). SbR56P was blocked at the plasma membrane in association with the endogenous plasma membrane t-SNARE due to an inhibition of SNARE complex dissociation, suggesting that the plasma membrane is its first target. Cell surface blockade of SbR56P could be rescued by coexpression of synaptophysin, a partner of Sb. Sb was blocked at the plasma membrane but SNARE complexes were unaffected in cells expressing defective dynamin, indicating that the phenotype of SbR56P was not due to an internalization defect. When expressed in neurons, SbR56P localized both to axonal and dendritic plasma membranes, showing that both domains are initial targets of Sb. The R56P mutation affects a highly conserved position in v-SNAREs, and might thus provide a general tool for identifying their first target membranes.

The proto-oncogene MYC has been implicated in the pathogenesis of many human cancers, including hematological and solid malignancies1. In the majority of T-ALL cases, MYC is aberrantly expressed downstream of activated NOTCH1 mutations. Despite the pivotal role of MYC in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and many other cancers, the mechanisms underlying MYC-mediated tumorigenesis remain inadequately understood2. Here we utilized a well-characterized zebrafish model of Myc-induced T-ALL for genetic studies to identify novel genes contributing to disease onset. We found that heterozygous inactivation of a tricarboxylic acid (TCA) cycle enzyme, dihydrolipoamide S-succinyltransferase (Dlst), significantly delayed tumor onset. Concordant with our zebrafish results, RNAi knockdown of DLST led to decreased cell viability and induction of apoptosis in human T-ALL cell lines. DLST is the E2 transferase of the -ketoglutarate dehydrogenase complex (KGDHC), which converts -ketoglutarate (-KG) to succinyl-CoA in the TCA cycle3. Polar metabolomics profiling revealed that the TCA cycle was disrupted by DLST knockdown in human T-ALL cells, as demonstrated by an accumulation of -KG and its precursor glutamine, as well as a loss of succinyl-CoA. Addition of succinate, the downstream TCA cycle intermediate, to human T-ALL cells was sufficient to rescue defects in cell viability caused by DLST inactivation. Taken together, our studies identify DLST as an important mediator of MYC-driven leukemogenesis and provide compelling evidence for the metabolic dependence of T-ALL cells on the TCA cycle. 1. Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999 May 13; 18(19): 3004-3016. 2. Sharma VM, Calvo JA, Draheim KM, Cunningham LA, Hermance N, Beverly L, et al. Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc. Molecular and cellular biology 2006 Nov; 26(21): 8022-8031. 3. Sheu KF, Blass JP. The alpha-ketoglutarate dehydrogenase complex. Ann N Y Acad Sci 1999; 893: 61-78. Citation Format: Nicole Anderson, Dun Li, H.L. Peng, Marc Mansour, Fabrice Laroche, Evisa Gjini, Daniel Helman, Itrat Harrold, Le Meng, Takaomi Sanda, Adam Amsterdam, Donna Neuberg, Travis Denton, Anurag Singh, A Thomas Look, Hui Feng. The TCA cycle transferase DLST is critical for MYC-mediated leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1180.

Efficient DNA delivery is essential for introducing new genes into living cells. However, effective virus-based systems carry risks and efficient synthetic systems that are non-toxic remain to be discovered. The bottle-neck in synthetic systems is cytotoxicity, caused by the high concentration of DNA-condensing compounds required for efficient uptake of DNA. Here we report a polyethyleneimine (PEI) grafted ultra-small graphene oxide (PEI-g-USGO) for transfection. By removing the free PEI and ensuring a high PEI density on small sized graphene, we obtained very high transfection efficiencies combined with very low cytotoxicity. Plasmid DNA could be transfected into mammalian cell lines with up to 95% efficiency and 90% viability. Transfection in zebrafish embryos was 90%, with high viability, compared to efficiencies of 30% or lower for established transfection technologies. This result suggests a novel approach to the design of synthetic gene delivery vehicles for research and therapy.

Cell metabolic processes are constantly producing reactive oxygen species (ROS), which have deleterious effects by triggering, for example, DNA damage. Numerous enzymes such as catalase, and small compounds such as vitamin C, provide protection against ROS. The TLDc domain of the human oxidation resistance protein has been shown to be able to protect DNA from oxidative stress; however, its mechanism of action is still not understood and no structural information is available on this domain. Structural information on the TLDc domain may therefore help in understanding exactly how it works. Here, the purification, crystallization and preliminary crystallographic studies of the TLDc domain from zebrafish are reported. Crystals belonging to the orthorhombic space group P2(1)2(1)2 were obtained and diffracted to 0.97 Å resolution. Selenomethionine-substituted protein could also be crystallized; these crystals diffracted to 1.1 Å resolution and the structure could be solved by SAD/MAD methods.

International audience; OBJECTIVES: Autism (MIM#209850) and schizophrenia (MIM#181500) are both neurodevelopmental psychiatric disorders characterized by a highly genetic component. Homeogenes and forkhead genes encode transcription factors, which have been involved in brain development and cell differentiation. Thus, they are relevant candidate genes for psychiatric disorders. Genetic studies have reported an association between autism and DLX2, HOXA1, EN2, ARX, and FOXP2 genes whereas only three studies of EN2, OTX2, and FOXP2 were performed on schizophrenia. Interestingly, most of these candidate genes contain trinucleotide repeats coding for polyamino acid stretch in which instability can be the cause of neurodevelopmental disorders. Our goal was to identify variations of coding trinucleotide repeats in schizophrenia, autism, and idiopathic mental retardation. METHODS: We screened the coding trinucleotide repeats of OTX1, EN1, DLX2, HOXA1, and FOXP2 genes in populations suffering from schizophrenia (247 patients), autism (98 patients), and idiopathic mental retardation (56 patients), and compared them with control populations (112 super controls and 202 healthy controls). RESULTS: Novel deletions and insertions of coding trinucleotide repeats were found in the DLX2, HOXA1, and FOXP2 genes. Most of these variations were detected in controls and no difference in their distribution was observed between patient and control groups. Two different polymorphisms in FOXP2 were, however, found only in autistic patients and the functional consequences of these variations of repeats have to be characterized and correlated to particular clinical features. CONCLUSION: This study did not identify specific disease risk variants of trinucleotide repeats in OTX1, EN1, DLX2, HOXA1, and FOXP2 candidate genes in neurodevelopmental psychiatric disorders.

Recent research has shown that ultra-small graphene oxide (USGO) particles functionalized with polyethylenimine (PEI) can be used to transfect human cell lines with very high efficiency and low toxicity. In this study, confocal fluorescence microscopy is used to provide more insight into the nature of the complexes formed by DNA and USGO_PEI and to track their localization during the transfection process. The results indicate that the DNA enters the nucleus in complex with the USGO_PEI, which remains inside the nucleus. Also, it is observed that USGO_PEI and plasmid DNA form large aggregates in the transfection medium. Both observations raise concerns that this type of nanomaterial might need further improvements for therapeutic use.