Terminal Deoxynucleotidyl Transferase Extension-Dominated In Situ Signal Attenuation-Free Electrochemical Platform and Its Logic Gate Manipulation

Signal generation of traditional electrochemical biosensors suffers from the random diffusion of electroactive probes in a electrolyte solution, which is accompanied by poor reaction kinetics and low signal stability from complex biological systems. Herein, a novel circuit system with autonomous compensation solution ohmic drop (noted as “fast-scan cyclic voltammetry (FSCV)”) is developed to solve the above problems, and employed to achieve terminal deoxynucleotide transferase (TdT) and its small molecule inhibitor analysis. At first, a typical TdT-mediated catalytic polymerization in the conditions of original DNA, deoxythymine triphosphate (dTTP) and Hg2+ is applied for the electrode assembly. The novel electrochemical method can provide some unattenuated signals due to in-situ Hg redox reaction, thus improving reaction kinetics and signal stability. This approach is mainly dependent on TdT-mediated reaction, so it can be applied properly for TdT investigation, and a detection limit of 0.067 U/mL (S/N=3) is achieved successfully. More interesting, we also mimic the function of TdT-related signal communication in various logic gates such as YES, NOT, AND, N-IMPLY, and AND-AND-N-IMPLY cascade circuit. This study provides a new method for the detection of TdT biomarkers in many types of diseases and the construction of a signal attenuation-free logic gate.

Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/.

Hexokinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera

Acetyl-CoA, the precursor of sex pheromone biosynthesis in Helicoverpa armigera, is generated from glycolysis. As the first speed-limited enzyme in glycolysis, Hexokinase (HK) plays an important role in acetyl-CoA production. However, the function of HK in sex pheromone production remains unclear. This study employed H. armigera as material to explore the role of HK in sex pheromone production. Results demonstrated that the transcription profile of HaHK in female moth pheromone glands (PGs) was consistent with the release fluctuation of sex pheromone. Interference of HaHK prevented the increase of acetyl-CoA content induced by PBAN. Therefore, knockdown of HaHK in female PGs caused significant decreases in (Z)-11-hexadecenal (Z11-16:Ald) production, female capability to attract males, and mating rate. Furthermore, sugar feeding (5% sugar) increased the transcription and enzymatic activity of HK. Pheromone biosynthesis activating neuropeptide (PBAN) signal phospho-activated HaHK in PGs and Sf9 cells via protein kinase A (PKA), as shown by pharmacological inhibitor analysis. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK, in turn promoted glycolysis to ensure the supply of acetyl-CoA, and finally facilitated sex pheromone biosynthesis and subsequent mating behavior.

Solving a Demand-Supply Mismatch by Enabling a Value Chain Orientation

Supply-Demand mismatch is a continuous challenge among suppliers creating poor customer service levels and often leading to higher costs to the entire supply chain, to meet the demands of the customer. This paper will highlight demand-supply mismatch issues between a steel supplier having supply issues with one of its premier automobile customers due to difficulties in forecasting the appropriate demand from the customer. The outcome of the original research was developing a framework for an integrated planning process that overlooks the entire demand planning and management of the customer, in addition to emphasizing the application of demand profiling that enabled to build a novel future state inventory model. In the context of the work published here, an enabler and inhibitor analysis was conducted, that studies the structure and processes within and between firms, to identify any business implications that may affect the demand-supply mismatch.

Context dependent isoform specific PI3K inhibition confers drug resistance in Hepatocellular carcinoma cells

BackgroundTargeted therapies for Primary liver cancer (HCC) is limited to the multi-kinase inhibitors, and not fully effective due to the resistance to these agents because of the heterogeneous molecular nature of HCC developed during chronic liver disease stages and cirrhosis. Although combinatorial therapy can increase the efficiency of targeted therapies through synergistic activities, isoform specific effects of the inhibitors are usually ignored. This study concentrated on PI3K/Akt/mTOR pathway and the differential combinatory bioactivities of isoform specific PI3K-α inhibitor (PIK-75) or PI3K-β inhibitor (TGX-221) with Sorafenib dependent on PTEN context.MethodsThe bioactivities of inhibitors on PTEN adequate Huh7 and deficient Mahlavu cells were investigated with real time cell growth, cell cycle and cell migration assays. Differentially expressed genes from RNA-Seq were identified by edgeR tool. Systems level network analysis of treatment specific pathways were performed with Prize Collecting Steiner Tree (PCST) on human interactome and enriched networks were visualized with Cytoscape platform.ResultsOur data from combinatory treatment of Sorafenib and PIK-75 and TGX-221 showed opposite effects; while PIK-75 displays synergistic effects on Huh7 cells leading to apoptotic cell death, Sorafenib with TGX-221 display antagonistic effects and significantly promotes cell growth in PTEN deficient Mahlavu cells. Transcriptomic states of combinatory treatments with PIK-75 and TGX-221 inhibitors were identified in PTEN deficient and adequate cells. Molecular interactions and cell signaling pathways were reconstructed and analyzed in-depth to understand mechanism of differential synergistic or antagonistic effects of PI3K-α (PIK-75) and PI3K-β (TGX-221) inhibitors with Sorafenib.ConclusionsSimultaneously constructed and analyzed differentially expressed cellular networks presented in this study, revealed distinct consequences of isoform specific PI3K inhibition in PTEN adequate and deficient liver cancer cells. We demonstrated the importance of context dependent and isoform specific PI3K/Akt/mTOR signaling inhibition in drug resistance during combination therapies. (https://github.com/cansyl/Isoform-spesific-PI3K-inhibitor-analysis).

Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

SummaryTumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. We reasoned that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Combining phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis identifies functional modules of phosphorylations. The majority of regulated phosphorylations could be assigned to an upstream kinase by inhibiting master kinases and spatial proteomics revealed phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovered a key role for transcriptional cyclin-dependent kinase (CDK) activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. Our comprehensive interrogation of TNF induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/.Highlights-Distinct phosphorylation events mark early and late TNF signaling-Inhibition of master kinases reveals TNF stimulation dependent kinase-substrate relations-TNF induces phosphorylation-dependent spatial rearrangement of hundreds of proteins-CDK kinase activity promotes TNF-induced cytokine expression and inhibits cell death-CDK12/13 inhibitors have potential as anti-inflammatory agents