Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma

Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety.To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection.Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications

A local mechanism mediates NAD-dependent protection of axon degeneration

Axon degeneration occurs frequently in neurodegenerative diseases and peripheral neuropathies. Important insight into the mechanisms of axon degeneration arose from findings that the degeneration of transected axons is delayed in Wallerian degeneration slow (Wlds) mice with the overexpression of a fusion protein with the nicotinamide adenine dinucleotide (NAD) synthetic enzyme, nicotinamide mononucleotide adenylyltransferase (Nmnat1). Although both Wlds and Nmnat1 themselves are functional in preventing axon degeneration in neuronal cultures, the underlying mechanism for Nmnat1- and NAD-mediated axon protection remains largely unclear. We demonstrate that NAD levels decrease in degenerating axons and that preventing this axonal NAD decline efficiently protects axons from degeneration. In support of a local protective mechanism, we show that the degeneration of axonal segments that have been separated from their soma could be prevented by the exogenous application of NAD or its precursor nicotinamide. Furthermore, we provide evidence that such Nmnat1/NAD-mediated protection is primarily mediated by their effects on local bioenergetics. Together, our results suggest a novel molecular pathway for axon degeneration

Alcohol promotes breast cancer cell invasion by regulating the Nm23-ITGA5 pathway

<p>Abstract</p> <p>Background</p> <p>Alcohol consumption is an established risk factor for breast cancer metastasis. Yet, the mechanism by which alcohol promotes breast cancer metastases is unknown. The ability of cancer cells to invade through tissue barriers (such as basement membrane and interstitial stroma) is an essential step towards establishing cancer metastasis. In the present study, we identify and examine the roles of two genes, <it>Nm23 </it>and <it>ITGA5</it>, in alcohol-induced breast cancer cell invasion.</p> <p>Methods</p> <p>Human breast cancer T47D cells were treated with ethanol at various concentrations. Boyden chamber invasion assays were used to measure cellular invasive ability. The mRNA expression level of metastasis suppressor genes including <it>Nm23 </it>was determined by qRT-PCR. <it>ITGA5 </it>was identified using a qRT-PCR array of 84 genes important for cell-cell and cell-extracellular matrix interactions. <it>Nm23 </it>overexpression in addition to <it>Nm23</it>- and <it>ITGA5 </it>knock-down were used to determine the role of the Nm23-ITGA5 pathway on cellular invasive ability of T47D cells. Protein expression levels were verified by Western blot.</p> <p>Results</p> <p>Alcohol increased the invasive ability of human breast cancer T47D cells in a dose-dependent manner through the suppression of the <it>Nm23 </it>metastatic suppressor gene. In turn, <it>Nm23 </it>down-regulation increased expression of fibronectin receptor subunit <it>ITGA5</it>, which subsequently led to increased cellular invasion. Moreover, <it>Nm23 </it>overexpression was effective in suppressing the effects of alcohol on cell invasion. In addition, we show that the effects of alcohol on invasion were also inhibited by knock-down of <it>ITGA5</it>.</p> <p>Conclusions</p> <p>Our results suggest that the Nm23-ITGA5 pathway plays a critical role in alcohol-induced breast cancer cell invasion. Thus, regulation of this pathway may potentially be used to prevent the establishment of alcohol-promoted metastases in human breast cancers.</p

10-qubit entanglement and parallel logic operations with a superconducting circuit

Here we report on the production and tomography of genuinely entangled Greenberger-Horne-Zeilinger states with up to 10 qubits connecting to a bus resonator in a superconducting circuit, where the resonator-mediated qubit-qubit interactions are used to controllably entangle multiple qubits and to operate on different pairs of qubits in parallel. The resulting 10-qubit density matrix is unambiguously probed, with a fidelity of $0.668 \pm 0.025$. Our results demonstrate the largest entanglement created so far in solid-state architectures, and pave the way to large-scale quantum computation.Comment: Revised version with 16 pages, 13 figures, and 2 table

Integrated transcriptome, small RNA and degradome sequencing approaches provide insights into Ascochyta blight resistance in chickpea

Ascochyta blight (AB) is one of the major biotic stresses known to limit the chickpea production worldwide. To dissect the complex mechanisms of AB resistance in chickpea, three approaches, namely, transcriptome, small RNA and degradome sequencing were used. The transcriptome sequencing of 20 samples including two resistant genotypes, two susceptible genotypes and one introgression line under control and stress conditions at two time points (3rd and 7th day post inoculation) identified a total of 6767 differentially expressed genes (DEGs). These DEGs were mainly related to pathogenesis�related proteins, disease resistance genes like NBS�LRR, cell wall biosynthesis and various secondary metabolite synthesis genes. The small RNA sequencing of the samples resulted in the identification of 651 miRNAs which included 478 known and 173 novel miRNAs. A total of 297 miRNAs were differentially expressed between different genotypes, conditions and time points. Using degradome sequencing and in silico approaches, 2131 targets were predicted for 629 miRNAs. The combined analysis of both small RNA and transcriptome datasets identified 12 miRNA�mRNA interaction pairs that exhibited contrasting expression in resistant and susceptible genotypes and also, a subset of genes that might be post�transcriptionally silenced during AB infection. The comprehensive integrated analysis in the study provides better insights into the transcriptome dynamics and regulatory network components associated with AB stress in chickpea and, also offers candidate genes for chickpea improvement

Developmental Context Determines Latency of MYC-Induced Tumorigenesis

One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis

Biophysical Studies of Bacterial Topoisomerases Substantiate Their Binding Modes to an Inhibitor

AbstractBacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G+) and Gram-negative (G−) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G+ strain (Streptococcus pneumoniae (sParE)) and a G− strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between β2 and α3, and the β2 and β6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents

Liquid-crystalline circularly polarised TADF emitters for high-efficiency, solution-processable organic light-emitting diodes

Achieving a high emission efficiency and a large luminescence asymmetry factor (glum) in a single molecule exhibiting circularly polarised thermally activated delayed fluorescence (CP-TADF) remains a formidable challenge. In this work, a proof-of-concept, liquid-crystalline CP-TADF molecule is proposed to realise high glum by taking advantage of the order inherent in liquid crystals. Employing a chiral dinaphthol-based CP-TADF molecule as the emissive unit, a pair of liquid-crystalline CP-TADF molecules (R/S-4) is synthesised via the introduction of six mesogenic moieties. The enantiomers show intense emission at about 520 nm which has clear TADF and liquid-crystalline characteristics. Both enantiomers display symmetrical electronic circular dichroism (CD) and circular polarisation luminescence (CPL) signals as thin films. Impressively, relatively large glum values of 0.11 are realised for the films. Solution-processed devices were fabricated using R/S-4 as the dopants, with the TADF molecule CzAcSF as the sensitiser. The OLEDs so prepared show a very high maximum external quantum efficiency of 21.2%, revealing a novel strategy for realising large glum values in CP-TADF

WldS Reduces Paraquat-Induced Cytotoxicity via SIRT1 in Non-Neuronal Cells by Attenuating the Depletion of NAD

WldS is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether WldS can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that WldS significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI), hydrogen peroxide, etoposide, tunicamycin or brefeldin A. WldS also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that WldS markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of WldS by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, WldS delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and WldS-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of WldS against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of WldS in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning