Controllable synthesis of MoS2@TiO2 nanocomposites for visual detection of dopamine secretion with highly-efficient enzymatic activity.

Dopamine (DA) plays an essential role in dopaminergic neuronal behavior and disease. However, current detection methods for discriminating the secretion of DA are hampered by the limitations of the requirement for bulky instrumentation and non-intuitive signals. Herein, we have controllably and proportionately integrated molybdenum disulfide (MoS2) with titanium dioxide (TiO2) to prepare MoS2@TiO2 nanocomposites (MoS2@TiO2 NCs) via a facile synthesis method. MoS2@TiO2 NCs with a certain reactant mass ratio have shown a significant enhancement in peroxidase-like activity with superiority of the nanocomposite structure compared to single MoS2 or natural enzyme. The method for catalyzing the decomposition of H2O2 by MoS2@TiO2 NCs and competition for hydroxyl radicals (˙OH) between the chromogenic agent and DA enable a sensitive, specific, and colorimetric DA analysis with a low detection limit of 0.194 µM and a wide linear detection range (0.8 to 100 µM). Because of the favorable detection performance, we were encouraged to explore and finally realize the visual detection of cellular DA secretion that is stimulated in a High-K+ neurocyte environment. Collectively, this method will provide a promising strategy for basic research in neuroscience with its portable, sensitive, and naked-eye detectable performance.

Establishment of a t(11;19), KMT2A Rearranged B-ALL Cell Line for Preclinical Evaluation and Novel Therapeutics Development for Refractory Infant Leukemia.

Leukemia, diagnosed in children less than 12 months of age, is a rare condition with an aggressive disease presentation and poor response to conventional chemotherapeutic agents. In addition, the unique vulnerability of the affected population does not always permit the use of markedly intense regimens with higher doses of cytotoxic agents. However, the unique biology of these leukemic cells also provides opportunities for the identification of effective and potentially well-tolerated targeted therapeutic strategies. In this report, we describe the establishment and characterization of a cell line from the blasts of an infant diagnosed with refractory B-cell acute lymphoblastic leukemia (ALL) carrying the characteristic histone lysine methyltransferase 2A (KMT2A) gene rearrangement. This cell line consists of rapidly proliferating clones of cells with chemosensitivity patterns previously described for KMT2A rearranged leukemia cells, including relative resistance to glucocorticoids and sensitivity to cytarabine. We also show effective targetability with menin inhibitors, indicating the activity of abnormal KMT2A-related pathways and the potential utility of this cell line in comprehensive drug library screens. Overall, our findings report the establishment and in vitro validation of a cell line for research into key aspects of infant leukemia biology and targeted therapeutics development.

Dual functionality of the antimicrobial agent taurolidine which demonstrates effective anti-tumor properties in pediatric neuroblastoma.

High-risk, relapsed and refractory neuroblastoma are associated with poor 5-years survival rates, demonstrating the need for investigational therapeutic agents to treat this disease. Taurolidine is derived from the aminosulfoacid taurine and has known anti-microbial and anti-inflammatory properties. Taurolidine has also demonstrated anti-neoplastic effects in a range of cancers, providing the rationale to investigate the activity of taurolidine against neuroblastoma in preclinical studies. We investigated the in vitro activity of taurolidine against neuroblastoma using the alamar blue cytotoxicity assay, phase-contrast light microscopy, western blotting and analysis of global gene expression by RNA-Seq. In vivo activity of taurolidine was evaluated using mouse xenograft models. In vitro pre-clinical data show that taurolidine is cytotoxic to neuroblastoma cell lines, inducing cell death by apoptosis. Analysis of global gene expression and determination of signaling pathway activation scores using the in silico Pathway Activation Network Decomposition Analysis (iPANDA) platform indicates that taurolidine has an effect on the Notch, mitogen-activated protein kinase (MAPK) and interleukin-10 (IL-10) signaling pathways. In vivo experiments in xenograft mouse models show that taurolidine decreases tumor growth and improves survival. These results provide supportive pre-clinical data on the activity of taurolidine against neuroblastoma. The findings support the rationale for further evaluation of taurolidine for the treatment of relapsed/refractory neuroblastoma patients in an early phase clinical trial.

Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

Sex steroid levels near the term of pregnancy do not alter lipopolysaccharide-induced fever in oophorectomized rats.

NEW FINDINGS: What is the central question of this study? Do plasma concentrations of oestrogen and progesterone similar to those observed near the term of pregnancy alter basal core temperature or the core temperature response to bacterial pyrogen in oophorectomized rats? What is the main finding and its importance? Plasma concentrations of oestrogen and progesterone similar to those observed near the term of pregnancy do not alter basal core temperature or the overall febrile response to bacterial pyrogen in oophorectomized rats. Thus, our data do not support the hypothesis that sex steroids mediate the regulated decrease in basal core temperature or the attenuated/absent core temperature response to bacterial pyrogen observed in rats near the term of pregnancy. Fever, an important component of the host's defence response to infection, is absent or attenuated in rats near the term of pregnancy concurrent with major changes in blood concentrations of the sex steroids, oestrogen and progesterone. The present experiments were carried out to determine the potential role of oestrogen and progesterone in mediating the altered core temperature response to bacterial pyrogen. For the experiments, oestrogen and progesterone were administered alone or in combination to oophorectomized, non-pregnant rats in concentrations that mimicked plasma levels of these hormones measured in pregnant rats on days 17, 18, 19 and 20 of gestation. Treatment with oestrogen or progesterone alone or in combination did not alter basal core temperature or the overall febrile response (i.e. 12 h fever index) to an EC50 dose of Escherichia coli lipopolysaccharide (i.e. 20 µg kg(-1) ). Administration of oestrogen did, however, influence the early core temperature response and increase the latency to fever following administration of lipopolysaccharide. Thus, our data provide evidence that although oestrogen may influence the early core temperature response to lipopolysaccharide, sex steroids in concentrations similar to those observed late in gestation do not alter the overall febrile response and are therefore unlikely to mediate the attenuated or absent febrile response to bacterial pyrogen in rats near the term of pregnancy.

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation.

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells.

Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8.

The myotubularins are a large family of inositol polyphosphate 3-phosphatases that, despite having common substrates, subsume unique functions in cells that are disparate. The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] at the D-3 position. Seven members are inactive because they lack the conserved cysteine residue in the CX(5)R motif required for activity. We studied a subfamily of homologous myotubularins, including myotubularin-related protein 6 (MTMR6), MTMR7, and MTMR8, all of which dimerize with the catalytically inactive MTMR9. Complex formation between the active myotubularins and MTMR9 increases their catalytic activity and alters their substrate specificity, wherein the MTMR6/R9 complex prefers PtdIns(3,5)P(2) as substrate; the MTMR8/R9 complex prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of MTMR6 toward PtdIns(3,5)P(2) by over 30-fold, and enhanced the activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 increased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns(3,5)P(2) and PtdIns(3)P, respectively. In cells, the MTMR6/R9 complex significantly increases the cellular levels of PtdIns(5)P, the product of PI(3,5)P(2) dephosphorylation, whereas the MTMR8/R9 complex reduces cellular PtdIns(3)P levels. Consequentially, the MTMR6/R9 complex serves to inhibit stress-induced apoptosis and the MTMR8/R9 complex inhibits autophagy.

Identification and In Vivo Validation of Unique Anti-Oncogenic Mechanisms Involving Protein Kinase Signaling and Autophagy Mediated by the Investigational Agent PV-10.

PV-10 is a 10% formulation of rose bengal sodium that has potent immunotherapeutic and anti-cancer activity against various tumors, including metastatic melanoma and refractory neuroblastoma. Currently, PV-10 is undergoing clinical testing for refractory metastatic neuroendocrine cancer and melanomas. However, preclinical investigation of PV-10 activity and its mechanisms against phenotypically and molecularly diverse adult solid tumors had not been conducted. In a panel of human cell lines derived from breast, colorectal, head and neck, and testicular cancers, we demonstrated that PV-10 induces cytotoxicity by apoptotic and autophagic pathways involving caspase-mediated PARP cleavage, downregulation of SQSTM1/p62, and upregulation of beclin-1. Treatment with PV-10 also consistently reduced phosphorylation of WNK1, which has been implicated in cancer cell migration and autophagy inhibition. By wound healing assay, PV-10 treatment inhibited the migration of cancer cells. Finally, significant inhibition of tumor growth was also noted in tumor-bearing mice treated with PV-10 by intralesional or systemic administration. In addition to known PV-10-mediated tumor-specific cytotoxic effects, we identified the mechanisms of PV-10 and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate clinical studies of PV-10 in the future.

The Small-Molecule E26-Transformation-Specific Inhibitor TK216 Attenuates the Oncogenic Properties of Pediatric Leukemia.

The E26-transformation-specific (ETS) transcription factors regulate multiple aspects of the normal hematopoietic system. There is an increasing body of evidence suggesting aberrant ETS activity and its contribution to leukemia initiation and progression. In this study, we evaluated the small-molecule ETS inhibitor TK216 and demonstrated its anti-tumor activity in pediatric leukemia. We found TK216 induced growth inhibition, cell cycle arrest and apoptosis and inhibited the migratory capability of leukemic cells, without significantly inhibiting the cell viability of normal blood mononuclear cells. Priming the leukemic cells with 5-Azacitidine enhanced the cytotoxic effects of TK216 on pediatric leukemia cells. Importantly, we found purine-rich box1 (PU.1) to be a potential target of TK216 in myeloid and B-lymphoid leukemic cells. In addition, TK216 sharply decreased Mcl-1 protein levels in a dose-dependent manner. Consistent with this, TK216 also potentiated the cytotoxic effects of Bcl-2 inhibition in venetoclax-resistant cells. The sustained survival benefit provided to leukemic cells in the presence of bone-marrow-derived conditioned media is also found to be modulated by TK216. Taken together, our data indicates that TK216 could be a promising targeted therapeutic agent for the treatment of acute myeloid and B-lymphoid leukemia.

Repeated dosing improves oncolytic rhabdovirus therapy in mice via interactions with intravascular monocytes.

There is debate in the field of oncolytic virus (OV) therapy, whether a single viral dose, or multiple administrations, is better for tumor control. Using intravital microscopy, we describe the fate of vesicular stomatitis virus (VSV) delivered systemically as a first or a second dose. Following primary administration, VSV binds to the endothelium, initiates tumor infection and activates a proinflammatory response. This initial OV dose induces neutrophil migration into the tumor and limits viral replication. OV administered as a second dose fails to infect the tumor and is captured by intravascular monocytes. Despite a lack of direct infection, this second viral dose, in a monocyte-dependent fashion, enhances and sustains infection by the first viral dose, promotes CD8 T cell recruitment, delays tumor growth and improves survival in multi-dosing OV therapy. Thus, repeated VSV dosing engages monocytes to post-condition the tumor microenvironment for improved infection and anticancer T cell responses. Understanding the complex interactions between the subsequent viral doses is crucial for improving the efficiency of OV therapy and virus-based vaccines.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

Human SNF5 arming of double-deleted vaccinia virus shows oncolytic and cytostatic activity against central nervous system atypical teratoid/rhabdoid tumor cells.

Central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is a rare, aggressive tumor that most often affects very young children. The common decisive molecular defect in AT/RT has been shown to be a single genetic alteration, i.e., the loss of hSNF5 gene that encodes for a subunit of the SWI/SNF complex that modulates chromatin remodeling activities. As a result, AT/RT cells display unregulated cell proliferation due to the dysfunction of an important epigenetic control. We have previously demonstrated the preclinical efficacy of the oncolytic double-deleted vaccinia virus (VVDD) against AT/RT. Here we report the establishment of a modified VVDD engineered to express wild type hSNF5 gene. We show that this reconstructed vaccinia virus retains comparable infectivity and in vitro cytotoxicity of the parent strain. However, in addition, hSNF5-arming of VVDD results in a decreased cell cycle S phase population and down-regulation of cyclin D1. These findings suggest that hSNF5-arming of VVDD may increase the efficacy in the treatment of AT/RT and validates, as a proof-of-concept, an experimental approach to enhance the effective use of novel modified oncolytic viruses in the treatment of tumors with loss of a tumor suppressor gene function.

Identification and in vitro validation of neoantigens for immune activation against high-risk pediatric leukemia cells.

There is experimental and clinical data to indicate the contribution of immune-escape mechanisms in relapsed/refractory pediatric leukemia. Studies have shown the accumulation of mutations that translate to peptides containing tumor-specific epitopes (neoantigens). The effectiveness of neoantigen-based vaccines has been shown in several clinical trials in adults. Though the initial results are encouraging, this knowledge must be developed to account for the uniqueness of pediatric cancer biology. We have completed the initial proof-of-concept analysis on a high-risk pediatric leukemia specimen and identified usable neoantigen sequences. We describe this approach, including the bioinformatics method and experimental model to verify their function that can be further broadened for personalized neoantigen prediction and testing for the generation of anticancer vaccines against high-risk pediatric leukemias.

Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase.

Strategies for assembly and analysis of human, yeast, and bacterial RNA polymerase elongation complexes are described, and methods are shown for millisecond phase kinetic analyses of elongation using rapid chemical quench flow. Human, yeast, and bacterial RNA polymerases function very similarly in NTP-Mg2+ commitment and phosphodiester bond formation. A "running start, two-bond, double-quench" protocol is described and its advantages discussed. These studies provide information about stable NTP-Mg2+ loading, phosphodiester bond synthesis, the processive transition between bonds, and sequence-specific effects on transcription elongation dynamics.

In Vitro Investigation Demonstrates IGFR/VEGFR Receptor Cross Talk and Potential of Combined Inhibition in Pediatric Central Nervous System Atypical Teratoid Rhabdoid Tumors.

BACKGROUND: Atypical teratoid rhabdoid tumor of the central nervous system (CNS ATRT) is a malignancy that commonly affects young children. The biological mechanisms contributing to tumor aggressiveness and resistance to conventional therapies in ATRT are unknown. Previous studies have shown the activity of insulin like growth factor-I receptor (IGF-1R) in ATRT tumor specimens and cell lines. IGF-1R has been shown to cross-talk with other receptor tyrosine kinases (RTKs) in a number of cancer types, leading to enhanced cell proliferation. OBJECTIVE: This study aims to evaluate the role of IGF-1 receptor cross-talk in ATRT biology and the potential for therapeutic targeting. METHODS: Cell lines derived from CNS ATRT specimens were analyzed for IGF-1 mediated cell proliferation. A comprehensive receptor tyrosine kinase (RTK) screen was conducted following IGF-1 stimulation. Bioinformatic analysis of publicly available cancer growth inhibition data to identify correlation between IC50 of a VEGFR inhibitor and IGF-1R expression. RESULTS: Comprehensive RTK screen identified VEGFR-2 cross-activation following IGF-1 stimulation. Bioinformatics analysis demonstrated a positive correlation between IC50 values of VEGFR inhibitor Axitinib and IGF-1R expression, supporting the critical influence of IGF-1R in modulating response to anti-angiogenic therapies. CONCLUSION: Overall, our data present a novel experimental framework to evaluate and utilize receptor cross-talk mechanisms to select effective drugs and combinations for future therapeutic trials in ATRT.

Alterations of Oral Microbiota in Chinese Patients With Esophageal Cancer.

Emerging evidence supports that oral microbiota are associated with health and diseases of the esophagus. How oral microbiota change in Chinese patients with esophageal cancer (EC) is unknown, neither is their biomarker role. For an objective to understand alterations of oral microbiota in Chinese EC patients, we conducted a case-control study including saliva samples from 39 EC patients and 51 healthy volunteers. 16S rDNA genes of V3-V4 variable regions were sequenced to identify taxon. Relationship between oral flora and disease was analyzed according to alpha diversity and beta diversity. Resultantly, the Shannon index (p = 0.2) and the Simpson diversity index (p = 0.071) were not significant between the two groups. Yet we still found several species different in abundance between the two groups. For the EC group, the most significantly increased taxa were Firmicutes, Negativicutes, Selenomonadales, Prevotellaceae, Prevotella, and Veillonellaceae, while the most significantly decreased taxa were Proteobacteria, Betaproteobacteria, Neisseriales, Neisseriaceae, and Neisseria. In conclusion, there are significant alterations in abundance of some oral microbiomes between the EC patients and the healthy controls in the studied Chinese participants, which may be meaningful for predicting the development of EC, and the potential roles of these species in EC development deserve further studies.

Potent in vitro and xenograft antitumor activity of a novel agent, PV-10, against relapsed and refractory neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial cancer in children. Although the prognosis for low-risk neuroblastoma patients is good, the 5-year survival rates for high-risk and relapsed patients are low. The poor survival rates for these patients demonstrate the need for novel therapeutic approaches to treat this disease. PV-10 is a sterile 10% solution of Rose Bengal that has previously been shown to induce cell death in a range of adult cancers, providing the rationale for studying the activity of PV-10 against neuroblastoma in preclinical studies. METHODS: The effects of PV-10 on neuroblastoma were investigated in vitro. Cytotoxicity assays were performed using the alamar blue assay on the following cell lines: SK-N-AS, SK-N-BE(2), IMR5, LAN1, SHEP, and SK-N-SH neuroblastoma cells, SK-N-MC neuroepithelioma cells, and normal primary, BJ, and WI38 fibroblasts. Phase-contrast, fluorescence, and time-lapse video microscopy; flow cytometry; and Western blotting were used to investigate the effects of PV-10 on SK-N-AS and IMR5 cells. Synergy with commonly used anticancer drugs was determined by calculation of combination indices in SK-N-AS and IMR5 cells. Mouse xenograft models of SK-N-AS and IMR5 tumors were also used to evaluate the efficacy of PV-10 in vivo. RESULTS: In vitro preclinical data demonstrate that pharmacologically relevant concentrations of PV-10 are cytotoxic to neuroblastoma cell lines. Studies to investigate target modulation in neuroblastoma cell lines show that PV-10 disrupts lysosomes, decreases the percentage of cells in S phase, and induces apoptosis in a concentration-, time-, and cell-line-dependent manner, and we also identify agents that are synergistic with PV-10. Furthermore, experiments in xenograft mouse models show that PV-10 induces tumor regression in vivo. CONCLUSION: Our study provides preclinical data on the efficacy of PV-10 against neuroblastoma and provides rationale for the development of an early phase clinical trial of this agent in relapsed and refractory neuroblastoma patients.

Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation.

The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the alpha1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in alpha1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 alpha1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.

Effects of hsa_circRBM23 on Hepatocellular Carcinoma Cell Viability and Migration as Produced by Regulating miR-138 Expression.

Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors. At present, the molecular mechanism of HCC remains unclear. A recent circular RNA (circRNA) profiling study showed that circRBM23 expression was upregulated in HCC tissues. Therefore, in this study, the impact of circRBM23 during the progression of HCC was evaluated. The expression levels of circRBM23 and miR-138 in HCC tissues and HCC cell lines were determined by RT-PCR and the results indicated that circRBM23 expression was increased in the HCC tissues and HCC cell lines, whereas miR-138 expression was decreased. An upregulation of circRBM23 expression in HCC cells was shown to increase cell viability, and also increased the ability of cells to migrate. Downregulation of circRBM23 was found to decrease cell viability, proliferation, and migration, and promote the expression of miR-138 and its related target genes, vimentin, and CCND3. Moreover, miR-138 was found to regulate HCC cell viability and migration, and the levels of vimentin and CCND3 protein expression were found to be inversely correlated with those of miR-138 expression. The downregulation of circRBM23 in HCC tissues can regulate the miR-138-mediated signal pathway by promoting miR-138 expression. The results in vivo demonstrated that circRBM23 is required for the tumorigenesis with downregulation of tumor suppressor miR-138. These data indicated that upregulated circRBM23 functioned as oncogene in HCC through regulating the tumor suppressor miR-138.

Visualizing Oncolytic Virus-Host Interactions in Live Mice Using Intravital Microscopy.

Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy.