A20 Prevents Inflammasome-Dependent Arthritis by Inhibiting Macrophage Necroptosis Through Its ZnF7 Ubiquitin-Binding Domain

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1β release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo

Microstructural Evolution in Thin Films of Electronic Materials

Contains reports on eight research projects and a list of publications.National Science FoundationU.S. Air Force - Office of Scientific ResearchJoint Services Electronics Program Contract DAAL03-89-C-0001IBM CorporationHitachi CorporationSemiconductor Research CorporationNational Institutes of Healt

Microstructural Evolution in Thin Films of Electronic Materials

Contains reports on five research projects and a list of publications and theses.Joint Services Electronics Program Contract DAAL03-89-C-0001Semiconductor Research CorporationNational Science FoundationU.S. Army Research OfficeHitachi LimitedIBM Corporatio

Mice with Different Susceptibility to Japanese Encephalitis Virus Infection Show Selective Neutralizing Antibody Response and Myeloid Cell Infectivity

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes public health problems in Asian countries. Only a limited number of JEV-infected individuals show symptoms and develop severe encephalitis, indicating host-dependent susceptibilities.C3H/HeN and DBA/2 mice, which exhibit different mortalities when infected by intraperitoneal inoculation with JEV, were used as experimental models to compare viral pathogenesis and host responses. One hundred infectious virus particles killed 95% of C3H/HeN mice whereas only 40% of DBA/2 mice died. JEV RNA was detected with similar low levels in peripheral lymphoid organs and in the sera of both mouse strains. High levels of viral and cytokine RNA were observed simultaneously in the brains of C3H/HeN and DBA/2 mice starting on days 6 and 9 post-infection, respectively. The kinetics of the cytokines in sera correlated with the viral replication in the brain. Significantly earlier and higher titers of neutralizing antibodies were detected in the DBA/2 strain. Primary embryonic fibroblasts, bone marrow-derived dendritic cells and macrophages from the two mouse strains were cultured. Fibroblasts displayed similar JEV replication abilities, whereas DBA/2-derived myeloid antigen-presenting cells had lower viral infectivity and production compared to the C3H/HeN–derived cells. may be elements associated with late and decreased mouse neuroinvasion

The Value of Intraoperative Parathyroid Hormone Monitoring in Localized Primary Hyperparathyroidism: A Cost Analysis

Minimally invasive parathyroidectomy (MIP) is the preferred approach to primary hyperparathyroidism (PHPT) when a single adenoma can be localized preoperatively. The added value of intraoperative parathyroid hormone (IOPTH) monitoring remains debated because its ability to prevent failed parathyroidectomy due to unrecognized multiple gland disease (MGD) must be balanced against assay-related costs. We used a decision tree and cost analysis model to examine IOPTH monitoring in localized PHPT. Literature review identified 17 studies involving 4,280 unique patients, permitting estimation of base case costs and probabilities. Sensitivity analyses were performed to evaluate the uncertainty of the assumptions associated with IOPTH monitoring and surgical outcomes. IOPTH cost, MGD rate, and reoperation cost were varied to evaluate potential cost savings from IOPTH. The base case assumption was that in well-localized PHPT, IOPTH monitoring would increase the success rate of MIP from 96.3 to 98.8%. The cost of IOPTH varied with operating room time used. IOPTH reduced overall treatment costs only when total assay-related costs fell below $110 per case. Inaccurate localization and high reoperation cost both independently increased the value of IOPTH monitoring. The IOPTH strategy was cost saving when the rate of unrecognized MGD exceeded 6$12,000 (compared with initial MIP cost of $3733). Setting the positive predictive value of IOPTH at 100% and reducing the false-negative rate to 0% did not substantially alter these findings. Institution-specific factors influence the value of IOPTH. In this model, IOPTH increased the cure rate marginally while incurring approximately 4% additional cost

A complete set of nascent transcription rates for yeast genes

The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) and the RNA pol ChIP-on-chip methods we generated a new, more accurate nascent transcription rate dataset. By comparing this dataset with the indirect ones obtained from the mRNA stabilities and mRNA amount datasets, we are able to obtain biological information about posttranscriptional regulation processes and a genomic snapshot of the location of the active transcriptional machinery. We have obtained nascent transcription rates for 4,670 yeast genes. The median RNA polymerase II density in the genes is 0.078 molecules/kb, which corresponds to an average of 0.096 molecules/gene. Most genes have transcription rates of between 2 and 30 mRNAs/hour and less than 1% of yeast genes have >1 RNA polymerase molecule/gene. Histone and ribosomal protein genes are the highest transcribed groups of genes and other than these exceptions the transcription of genes is an infrequent phenomenon in a yeast cell

Endothelial Surface Layer Degradation by Chronic Hyaluronidase Infusion Induces Proteinuria in Apolipoprotein E-Deficient Mice

Functional studies show that disruption of endothelial surface layer (ESL) is accompanied by enhanced sensitivity of the vasculature towards atherogenic stimuli. However, relevance of ESL disruption as causal mechanism for vascular dysfunction remains to be demonstrated. We examined if loss of ESL through enzymatic degradation would affect vascular barrier properties in an atherogenic model. Eight week old male apolipoprotein E deficient mice on Western-type diet for 10 weeks received continuous active or heat-inactivated hyaluronidase (10 U/hr, i.v.) through an osmotic minipump during 4 weeks. Blood chemistry and anatomic changes in both macrovasculature and kidneys were examined. Infusion with active hyaluronidase resulted in decreased ESL (0.32±0.22 mL) and plasma volume (1.03±0.18 mL) compared to inactivated hyaluronidase (0.52±0.29 mL and 1.28±0.08 mL, p<0.05 respectively).Active hyaluronidase increased proteinuria compared to inactive hyaluronidase (0.27±0.02 vs. 0.15±0.01 µg/µg protein/creatinin, p<0.05) without changes in glomerular morphology or development of tubulo-interstitial inflammation. Atherosclerotic lesions in the aortic branches showed increased matrix production (collagen, 32±5 vs. 18±3%; glycosaminoglycans, 11±5 vs. 0.1±0.01%, active vs. inactive hyaluronidase, p<0.05). ESL degradation in apoE deficient mice contributes to reduced increased urinary protein excretion without significant changes in renal morphology. Second, the induction of compositional changes in atherogenic plaques by hyaluronidase point towards increased plaque vulnerability. These findings support further efforts to evaluate whether ESL restoration is a valuable target to prevent (micro) vascular disease progressio

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions

Drug Discovery Using Chemical Systems Biology: Identification of the Protein-Ligand Binding Network To Explain the Side Effects of CETP Inhibitors

Systematic identification of protein-drug interaction networks is crucial to correlate complex modes of drug action to clinical indications. We introduce a novel computational strategy to identify protein-ligand binding profiles on a genome-wide scale and apply it to elucidating the molecular mechanisms associated with the adverse drug effects of Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors are a new class of preventive therapies for the treatment of cardiovascular disease. However, clinical studies indicated that one CETP inhibitor, Torcetrapib, has deadly off-target effects as a result of hypertension, and hence it has been withdrawn from phase III clinical trials. We have identified a panel of off-targets for Torcetrapib and other CETP inhibitors from the human structural genome and map those targets to biological pathways via the literature. The predicted protein-ligand network is consistent with experimental results from multiple sources and reveals that the side-effect of CETP inhibitors is modulated through the combinatorial control of multiple interconnected pathways. Given that combinatorial control is a common phenomenon observed in many biological processes, our findings suggest that adverse drug effects might be minimized by fine-tuning multiple off-target interactions using single or multiple therapies. This work extends the scope of chemogenomics approaches and exemplifies the role that systems biology has in the future of drug discovery