Increased Efficacy of Histone Methyltransferase G9a Inhibitors Against <i>MYCN</i>-Amplified Neuroblastoma.

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a pediatric solid tumor with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumors is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638, and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumor suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2, and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumors

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5

Non-invasive fractional flow reserve (FFRCT) in the evaluation of acute chest pain ? Concepts and first experiences

Objective: To evaluate 30 day rate of major adverse cardiac events (MACE) utilizing cCTA and FFRCT for evaluation of patients presenting to the Emergency Department (ED) with acute chest pain. Materials and methods: Patients between the ages of 18?95 years who underwent clinically indicated cCTA and FFRCT in the evaluation of acute chest pain in the emergency department were retrospectively evaluated for 30 day MACE, repeat presentation/admission for chest pain, revascularization, and additional testing. Results: A total of 59 patients underwent CCTA and subsequent FFRCT for the evaluation of acute chest pain in the ED over the enrollment period. 32 out of 59 patients (54 %) had negative FFRCT (>0.80) out of whom 18 patients (55 %) were discharged from the ED. Out of the 32 patients without functionally significant CAD by FFRCT, 32 patients (100 %) underwent no revascularization and 32 patients (100 %) had no MACE at the 30-day follow-up period. Conclusion: In this limited retrospective study, patients presenting to the ED with acute chest pain and with CCTA with subsequent FFRCT of >0.8 had no MACE at 30 days; however, for many of these patients results were not available at time of clinical decision making by the ED physician

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene

Correction: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

Present: The originally supplied Figure 5 contains duplicate total-ERK panels. Correct: The proper Figure 5 appears below. The authors sincerely apologize for this error

Ukupno harmoničko izobličenje i brzina prostorne modalne informacije za analizu haptičkog paralelnog gibanja

In this paper, two kinds of evaluation index for the haptic motion analysis in parallel multiple degrees–of–freedom (MDOF) system are proposed. At first, the spatial modal decomposition method based on discrete Fourier series expansion (DFS) is presented. Spatial modal information expresses a motion element that corresponds to a specific physical action. The spatial modal information can mathematically be defined by the Fourier coefficients. Then, this paper proposes the total harmonic distortion (THD) and the content rate of the haptic modal information as motion evaluation indexes. THD of the spatial modal information can evaluate the complexity of the human motion and/or the deformability of the contact environment. Content rate of the spatial modal information can evaluate the priority of motion element. Some experimental results on the bilateral motion control of a parallel five DOF haptic system are shown, in order to confirm the utility of the proposed indexes.U ovom radu predložena su dva indikatora vrednovanja haptičkog gibanja u paralelnom sustavu s više stupnjeva slobode. Prikazana je metoda prostorne modalne dekompozicije zasnovana na proširenom diskretnom Fourierovom redu. Prostorna modalna informacija predstavlja element koji odgovara specifičnoj fizikalnoj radnji. Prostorna modalna informacija matematički se može opisati koristeći Fourierove koeficijente. U ovom se radu kao indikatori za evaluaciju gibanja predlažu ukupno harmoničko izobličenje i brzina haptičke modalne informacije. Ukupnim harmoničkim izobličenjem prostorne modalne informacije može se ocijeniti kompleksnost ljudskog gibanja i/ili deformabilnost kontaktne okoline. Przina prostorne modalne informacije ocjenjuje prioritet elementa u gibanju. Kako bi se potvrdila korisnost predloženih indikatora vrednovanja prikazani su eksperimentalni rezultati dobiveni dvoosnim prostornim upravljanjem paralelnim haptičkim sustavom s pet stupnjeva slobode

The Feasibility, Tolerability, Safety, and Accuracy of Low-radiation Dynamic Computed Tomography Myocardial Perfusion Imaging With Regadenoson Compared With Single-photon Emission Computed Tomography

Objectives: Computed tomography (CT) myocardial perfusion imaging (CT-MPI) with hyperemia induced by regadenoson was evaluated for the detection of myocardial ischemia, safety, relative radiation exposure, and patient experience compared with single-photon emission computed tomography (SPECT) imaging. Materials and Methods: Twenty-four patients (66.5 y, 29% male) who had undergone clinically indicated SPECT imaging and provided written informed consent were included in this phase II, IRB-approved, and FDA-approved clinical trial. All patients underwent coronary CT angiography and CT-MPI with hyperemia induced by the intravenous administration of regadenoson (0.4 mg/5 mL). Patient experience and findings on CT-MPI images were compared to SPECT imaging. Results: Patient experience and safety were similar between CT-MPI and SPECT procedures and no serious adverse events due to the administration of regadenoson occurred. SPECT resulted in a higher number of mild adverse events than CT-MPI. Patient radiation exposure was similar during the combined coronary computed tomography angiography and CT-MPI (4.4 [2.7] mSv) and SPECT imaging (5.6 [1.7] mSv) (P-value 0.401) procedures. Using SPECT as the reference standard, CT-MPI analysis showed a sensitivity of 58.3% (95% confidence interval [CI]: 27.7-84.8), a specificity of 100% (95% CI: 73.5-100), and an accuracy of 79.1% (95% CI: 57.9-92.87). Low apparent sensitivity occurred when the SPECT defects were small and highly suspicious for artifacts. Conclusions: This study demonstrated that CT-MPI is safe, well tolerated, and can be performed with comparable radiation exposure to SPECT. CT-MPI has the benefit of providing both complete anatomic coronary evaluation and assessment of myocardial perfusion

Accuracy of an Artificial Intelligence Deep Learning Algorithm Implementing a Recurrent Neural Network With Long Short-term Memory for the Automated Detection of Calcified Plaques From Coronary Computed Tomography Angiography

Purpose: The purpose of this study was to evaluate the accuracy of a novel fully automated deep learning (DL) algorithm implementing a recurrent neural network (RNN) with long short-term memory (LSTM) for the detection of coronary artery calcium (CAC) from coronary computed tomography angiography (CCTA) data. Materials and Methods: Under an IRB waiver and in HIPAA compliance, a total of 194 patients who had undergone CCTA were retrospectively included. Two observers independently evaluated the image quality and recorded the presence of CAC in the right (RCA), the combination of left main and left anterior descending (LM-LAD), and left circumflex (LCx) coronary arteries. Noncontrast CACS scans were allowed to be used in cases of uncertainty. Heart and coronary artery centerline detection and labeling were automatically performed. Presence of CAC was assessed by a RNN-LSTM. The algorithm's overall and per-vessel sensitivity, specificity, and diagnostic accuracy were calculated. Results: CAC was absent in 84 and present in 110 patients. As regards CCTA, the median subjective image quality, signal-to-noise ratio, and contrast-to-noise ratio were 3.0, 13.0, and 11.4. A total of 565 vessels were evaluated. On a per-vessel basis, the algorithm achieved a sensitivity, specificity, and diagnostic accuracy of 93.1% (confidence interval [CI], 84.3%-96.7%), 82.76% (CI, 74.6%-89.4%), and 86.7% (CI, 76.8%-87.9%), respectively, for the RCA, 93.1% (CI, 86.4%-97.7%), 95.5% (CI, 88.77%-98.75%), and 94.2% (CI. 90.2%-94.6%), respectively, for the LM-LAD, and 89.9% (CI, 80.2%-95.8%), 90.0% (CI, 83.2%-94.7%), and 89.9% (CI, 85.0%-94.1%), respectively, for the LCx. The overall sensitivity, specificity, and diagnostic accuracy were 92.1% (CI, 92.1%-95.2%), 88.9% (CI. 84.9%-92.1%), and 90.3% (CI, 88.0%-90.0%), respectively. When accounting for image quality, the algorithm achieved a sensitivity, specificity, and diagnostic accuracy of 76.2%, 87.5%, and 82.2%, respectively, for poor-quality data sets and 93.3%, 89.2% and 90.9%, respectively, when data sets rated adequate or higher were combined. Conclusion: The proposed RNN-LSTM demonstrated high diagnostic accuracy for the detection of CAC from CCTA

Development of a multiplex DNA-based traceability tool for crop plant materials

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project ‘Tracing the origin of food’ (TRACE), a DNA-based multiplex detection tool was developed—the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide