MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is ...maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN-PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN-PA2G4 protein-protein interface had potent inhibitory effects on neuroblastoma tumorigenesis
. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. SIGNIFICANCE: Competitive chemical inhibition of the PA2G4-MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.
Course‐based Undergraduate Research Experiences (CUREs) offer an inclusive means to engage students in the scientific process and enhance student learning gains and persistence in STEM. To realize ...the benefits of CURE implementation, is there a minimum length of CURE? We studied the learning and attitudinal outcomes of more than 1,000 undergraduate students across the United States involved in chemistry, biochemistry, and molecular biology CUREs using malate dehydrogenase as the model system. There were three conditions, complete semester CURE (cCURE), modular CURE of about six weeks in length embedded into a laboratory course (mCURE), and no CURE (control). We also looked at the impact these conditions had on students who are persons excluded from STEM due to ethnicity or race (PEER). We hypothesized that the longer the students spent in a CURE the better their outcomes. We measured student outcomes using several validated measures and compared the conditions using ANOVA, ANCOVA, and chi‐square analyses. We found cCURE students had higher experimental design learning and STEM support than control students. We found cCURE>mCURE>control for student’s report of their interest in conducting research in the future, support for STEM students, and their interest in a STEM career. Students in the cCURE and control had higher positive attitudes about scientific research than students in the mCURE. There were no differences between conditions for negative attitudes towards science, science literacy, and beliefs about learning science. We found PEER students reporting the same results as White/Asian students, except for their interest in conducting research: PEER students in mCUREs had higher interest than their White/Asian counterparts in conducting research. We conclude that the overall pattern was for students in the cCURE condition to have better learning and attitudinal outcomes than students in the mCURE and control conditions. Additionally, PEER student outcomes in CURE conditions were similar to their White/Asian counterparts. These results should encourage faculty to use CUREs in laboratory courses to improve all students’ outcomes and indicate that even a short CURE embedded within a traditional laboratory course can benefit students.
Relapse rates among individuals with substance use disorder (SUD) remain high and new treatment approaches are needed, which require evaluation in randomized controlled trials (RCTs). Measurement and ...interpretation challenges for SUD RCT data are often ignored or presented only in statistical analysis plans. Since different analytic approaches may result in different estimates and thus interpretations of the treatment effect, it is important to present this clearly throughout the trial. Inconsistencies between study analyses and objectives present further challenges for interpretation and cross-study comparisons. The recent International Council for Harmonization (ICH) addendum provides standardized language and a common framework for aligning trial objectives, design, conduct, and analysis. The framework focuses on estimands, which describe the treatment effect and link the trial objective with the scientific question and the analytic approach. The use of estimands offers SUD researchers and clinicians the opportunity to explicitly address events that affect measurement and interpretation at the outset of the trial. Furthermore, the use of standard terminology can lead to clearer interpretations of SUD trials and the treatments evaluated in SUD trials. Resources for understanding and applying estimands are needed to optimize the use of this new, helpful framework. This Perspective provides this resource for SUD researchers. Specifically, it highlights the relevance of estimands for SUD trials. Furthermore, it demonstrates how estimands can be used to develop clinically relevant analyses to address challenges in SUD trials. It also shows how a standardized framework can be employed to improve the interpretation and presentation of SUD study findings.
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Dostopno za:
DOBA, FSPLJ, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ...ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumonia e infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro . NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation–induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro , cultured ErbB4 −/− ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii –induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC.
ABSTRACT
Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with ...responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR‐3, CAOV‐3; patient samples malignant 1 and 2) and resistant (DOV‐13, SKOV‐3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70‐fold with malignant 2, 43‐fold with OVCAR‐3). Using agonists, antagonists, and shRNA‐mediated knockdown of dsRNA receptors, we show that TLR3, RIG‐I, and mda5 coordinated a caspase 8/9‐ and interferon‐dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5‐to 7.5‐fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4–0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI= 1.5–2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA‐sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.—Van, D. N., Roberts, C. F., Marion, J. D., Lépine, S., Harikumar, K. B., Schreiter, J., Dumur, C. I., Fang, X., Spiegel, S., Bell, J. K. Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics. FASEB J. 26, 3188–3198 (2012). www.fasebj.org
The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to ...be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.
Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. ...We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.
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► Structure of quaternary complex of TLR3ecd with three neutralizing antibodies. ► Lateral clustering of TLR3:dsRNA SUs required for TLR3 signaling. ► Antibodies neutralize by blocking SU formation or lateral clustering.
The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP ...phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R145 of the PilZ domain motif, R145xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions.
Binding of c-di-GMP to its Borrelia receptor proteins triggers a structural rearrangement that may serve as an on/off switch for niche specific effector functions.
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