Multivariate Perturbation of a Growth Factor-Cytokine Signalling Network Reveals Complex Systemic Responses in Glioblastoma Cells

Glioblastoma cells can evade TRAIL-induced apoptosis through various strategies involving the growth factor-activated MEK–MAPK/ERK and PI3K–Akt/PKB pro-survival signalling cascades. Although these signalling cascades have been studied extensively, our understanding of how they interact and participate in modulating apoptosis as part of a dynamic cell-wide network of signalling proteins is limited by traditional univariate experimental paradigms. Here, we study three human glioblastoma cell lines with differential response to TRAIL-induced apoptosis: LN229 (resistant), T98G, and A172 (both susceptible). We show that differential TRAIL susceptibility in these cell lines is unrelated to expression levels of agonist (DR4 and DR5) or antagonist (DcR1, DcR2, and OPG) receptors for TRAIL and thus TRAIL-induced apoptosis in these cell lines is modulated at the intracellular signalling level. Serum, comprising multiple factors that regulate cellular activity, enhances TRAIL resistance in T98G but not LN229 and A172 cell lines. This protective effect against TRAIL-induced apoptosis is recapitulated by the prototypical survival factor PDGF in T98G cells. Univariate inhibition of cell survival signalling cascades with MEK inhibitor U0126 and PI3K inhibitor LY294002 sensitized T98G cells to TRAIL but did not abrogate PDGF-mediated protection. However, further perturbation with inhibitors in a combinatorial and multivariate manner reveal synergistic effects and complex systemic responses which may be a basis for uncovering novel insights into the regulation of TRAIL-induced apoptosis.Singapore-MIT Alliance (SMA

Design, Fabrication and Functional Analysis of a New Protein Array Based on ssDNA-based Assembly

In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction.Singapore-MIT Alliance (SMA

DNA Directed Assembly Probe for Detecting DNA-Protein Interaction in Microarray Format

Quantifying DNA-protein interaction using DNA microarrays are gaining increasing attention due to their ability to profile specificity of interactions in a high-throughput manner. This paper describes a new approach that used the ability of ssDNA-dsDNA probe to complex with DNA binding proteins in the solution phase and then spatially immobilized onto microarray through specific DNA hybridization. In one case, the Spatially Addressable DNA Array (SADA) approach demonstrated that enzymatic cleavage in solution is more efficient than if conducted heterogeneously. In addition, binding of RNA polymerase with promoter DNA could be detected with this strategy.Singapore-MIT Alliance (SMA

Mitochondrial Localized STAT3 Is Involved in NGF Induced Neurite Outgrowth

Background: Signal transducer and activator of transcription 3 (STAT3) plays critical roles in neural development and is increasingly recognized as a major mediator of injury response in the nervous system. Cytokines and growth factors are known to phosphorylate STAT3 at tyrosine 705 with or without the concomitant phosphorylation at serine 727, resulting in the nuclear localization of STAT3 and subsequent transcriptional activation of genes. Recent evidence suggests that STAT3 may control cell function via alternative mechanisms independent of its transcriptional activity. Currently, the involvement of STAT3 mono-phosphorylated at residue serine 727 (P-Ser-STAT3) in neurite outgrowth and the underlying mechanism is largely unknown. Principal Findings: In this study, we investigated the role of nerve growth factor (NGF) induced P-Ser-STAT3 in mediating neurite outgrowth. NGF induced the phosphorylation of residue serine 727 but not tyrosine 705 of STAT3 in PC12 and primary cortical neuronal cells. In PC12 cells, serine but not tyrosine dominant negative mutant of STAT3 was found to impair NGF induced neurite outgrowth. Unexpectedly, NGF induced P-Ser-STAT3 was localized to the mitochondria but not in the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NGF induced neurite outgrowth and the production of reactive oxygen species (ROS). Conclusion: Taken together, the findings herein demonstrated a hitherto unrecognized novel transcription independen

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Nonaudit services purchases, audit committee effectiveness and investment opportunity set.

This study examines the extent to which effective audit committees limit the level of nonaudit services purchased from the firms’ external auditors. We hypothesize that thenegative association between audit committee effectiveness and nonaudit services purchases is moderated by the investment opportunity set of the firm

Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

Abstract The preservation of nucleic acids from clinical samples is critical to facilitate accurate molecular diagnosis. The use of a paper matrix, Flinders Technology Associates (FTA) Elute cards, to archive DNA and viral RNA is well-documented. However, the feasibility of FTA Elute cards for archiving serum and serum exosomal microRNAs (miRNAs) remains unclear. Here, we performed a comprehensive evaluation of FTA Elute cards for miRNA storage and recovery in different pre-analytical conditions. The recovery of serum miRNA dry-spotted on FTA Elute cards by direct elution with water at high temperature was poor. However, serum miRNAs dry-spotted on the cards were isolated with about 40% yield when using QIAzol lysis reagent and recovery was improved remarkably (>80%) upon extraction from cards pre-treated with trehalose. miRNAs stored on the cards remained stable at room temperature and can be kept for prolonged periods. Furthermore, miRNAs could be similarly recovered from serum exosomes dry-spotted on the cards. Importantly, when using sera from gastric cancer (GC) patients, the miRNAs were efficiently recovered from trehalose pre-treated cards without affecting their representation. Collectively, we have demonstrated the potential of FTA Elute cards to archive serum and serum exosomal miRNAs, making it useful for biomarker discovery and diagnostics

Concise synthesis and two-photon-excited deep-blue emission of 1,8-diazapyrenes

Efficient violet–blue-emitting molecules are especially useful for applications in full-color displays, solid-state lighting, as well as in two-photon absorption (TPA) excited frequency-upconverted violet–blue lasing. However, the reported violet–blue-emitting molecules generally possess small TPA cross sections. In this work, new 1,8-diazapyrenes derivatives 3 with blue two-photon-excited fluorescence emission were concisely synthesized by the coupling reaction of readily available 1,4-naphthoquinone O,O-diacetyl dioxime (1) with internal alkynes 2 under the [{RhCl2Cp*}2]–Cu(OAc)2 (Cp*=pentamethylcyclopentadienyl ligand) bimetallic catalytic system. Elongation of the π-conjugated length of 1,8-diazapyrenes 3 led to the increase of TPA cross sections without the expense of a redshift of the emission wavelength, probably due to the rigid planar structure of chromophores. It is especially noteworthy that 2,3,6,7-tetra(4-bromophenyl)-1,8-diazapyrene (3c) has a larger TPA cross section than those of other molecules reported so far. These experimental results are explained in terms of the effects of extension of the π-conjugated system, intramolecular charge transfer, and reduced detuning energy