Genome-wide genetic screen identifies host ubiquitination as important for Legionella pneumophila Dot/Icm effector translocation.

The Dot/Icm system of Legionella pneumophila is essential for virulence and delivers a large repertoire of effectors into infected host cells to create the Legionella containing vacuole. Since the secretion of effectors via the Dot/Icm system does not occur in the absence of host cells, we hypothesised that host factors actively participate in Dot/Icm effector translocation. Here we employed a high-throughput, genome-wide siRNA screen to systematically test the effect of silencing 18,120 human genes on translocation of the Dot/Icm effector, RalF, into HeLa cells. For the primary screen, we found that silencing of 119 genes led to increased translocation of RalF, while silencing of 321 genes resulted in decreased translocation. Following secondary screening, 70 genes were successfully validated as 'high confidence' targets. Gene set enrichment analysis of siRNAs leading to decreased RalF translocation, showed that ubiquitination was the most highly overrepresented category in the pathway analysis. We further showed that two host factors, the E2 ubiquitin-conjugating enzyme, UBE2E1, and the E3 ubiquitin ligase, CUL7, were important for supporting Dot/Icm translocation and L. pneumophila intracellular replication. In summary, we identified host ubiquitin pathways as important for the efficiency of Dot/Icm effector translocation by L. pneumophila, suggesting that host-derived ubiquitin-conjugating enzymes and ubiquitin ligases participate in the translocation of Legionella effector proteins and influence intracellular persistence and survival.

Genes regulating membrane-associated E-cadherin and proliferation in adenomatous polyposis coli mutant colon cancer cells: High content siRNA screen.

Truncating mutations in the tumour suppressor gene APC occur frequently in colorectal cancers and result in the deregulation of Wnt signalling as well as changes in cell-cell adhesion. Using quantitative imaging based on the detection of membrane-associated E-cadherin, we undertook a protein coding genome-wide siRNA screen to identify genes that regulate cell surface E-cadherin in the APC-defective colorectal cancer cell line SW480. We identified a diverse set of regulators of E-cadherin that offer new insights into the regulation of cell-cell adhesion, junction formation and genes that regulate proliferation or survival of SW480 cells. Among the genes whose depletion promotes membrane-associated E-cadherin, we identified ZEB1, the microRNA200 family, and proteins such as a ubiquitin ligase UBE2E3, CDK8, sorting nexin 27 (SNX27) and the matrix metalloproteinases, MMP14 and MMP19. The screen also identified 167 proteins required for maintaining E-cadherin at cell-cell adherens junctions, including known junctional proteins, CTNND1 and CTNNA1, as well as signalling enzymes, DUSP4 and MARK2, and transcription factors, TEAD3, RUNX2 and TRAM2. A better understanding of the post-translational regulation of E-cadherin provides new opportunities for restoring cell-cell adhesion in APC-defective cells.

A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

Baseline Analysis of a Young α-1-Antitrypsin Deficiency Liver Disease Cohort Reveals Frequent Portal Hypertension.

OBJECTIVES: α-1-Antitrypsin (A1AT) deficiency is a common genetic disease with an unpredictable and highly variable course. The Childhood Liver Disease Research and Education Network is a National Institutes of Health, multicenter, longitudinal consortium studying pediatric liver diseases, with the objective of prospectively defining natural history and identifying disease modifiers. METHODS: Longitudinal, cohort study of A1AT patients' birth through 25 years diagnosed as having liver disease, type PIZZ or PISZ. Medical history, physical examination, laboratory, imaging, and standardized survey tool data were collected during the provision of standard of care. RESULTS: In the present report of the cohort at baseline, 269 subjects were enrolled between November 2008 and October 2012 (208 with their native livers and 61 postliver transplant). Subjects with mild disease (native livers and no portal hypertension [PHT]) compared to severe disease (with PHT or postliver transplant) were not different in age at presentation. A total of 57% of subjects with mild disease and 76% with severe disease were jaundiced at presentation (P = 0.0024). A total of 29% of subjects with native livers had PHT, but age at diagnosis and growth were not different between the no-PHT and PHT groups (P > 0.05). Subjects with native livers and PHT were more likely to have elevated bilirubin, ALT, AST, INR, and GGTP than the no-PHT group (P << 0.001), but overlap was large. Chemistries alone could not identify PHT. CONCLUSIONS: Many subjects with A1AT presenting with elevated liver tests and jaundice improve spontaneously. Subjects with PHT have few symptoms and normal growth. Longitudinal cohort follow-up will identify genetic and environmental disease modifiers.

Functional genomics down under: RNAi screening in the Victorian Centre for Functional Genomics.

The Victorian Centre for Functional Genomics (VCFG) is an RNAi screening facility housed at the Peter MacCallum Cancer Centre in Melbourne, Australia. The Peter Mac is Australia's largest dedicated Cancer Research Institute, home to a team of over 520 scientists that focus on understanding the genetic risk of cancer, the molecular events regulating cancer growth and dissemination and improving detection through new diagnostic tools (www.petermac.org). Peter Mac is a well recognised technology leader and established the VCFG with a view to enabling researchers Australia and New Zealand-wide access to cutting edge functional genomics technology, infrastructure and expertise. This review documents the technology platforms operated within the VCFG and provides insight into the workflows and analysis pipelines currently in operation.

A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation.

The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.

Nanotechnology and glaucoma: little particles for a big disease.

PURPOSE OF REVIEW: Current medical treatments designed to halt the progressive loss of retinal ganglion cells (RGCs) in glaucoma are limited by low bioavailability to target tissues and lack of patient adherence to frequent dosing regimens. For a certain percentage of patients with glaucoma, reducing intraocular pressure (IOP) does not stop disease progression, motivating the search for new therapeutic targets and delivery systems. RECENT FINDINGS: The emerging science of nanoparticles has the potential to address the current limitations of glaucoma therapy by improving drug bioavailability, exploiting IOP-independent targets such as RGC neuroprotection, and optimizing gene therapy as a more permanent treatment for glaucoma. SUMMARY: We review the recent advances in nanoparticle-based glaucoma therapy with a focus on drug delivery to the eye, as well as novel applications including gene therapy.

Acute liver failure in children: the first 348 patients in the pediatric acute liver failure study group.

OBJECTIVES: To determine short-term outcome for children with acute liver failure (ALF) as it relates to cause, clinical status, and patient demographics and to determine prognostic factors. STUDY DESIGN: A prospective, multicenter case study collecting demographic, clinical, laboratory, and short-term outcome data on children from birth to 18 years with ALF. Patients without encephalopathy were included if the prothrombin time and international normalized ratio remained > or = 20 seconds and/or >2, respectively, despite vitamin K. Primary outcome measures 3 weeks after study entry were death, death after transplantation, alive with native liver, and alive with transplanted organ. RESULTS: The cause of ALF in 348 children included acute acetaminophen toxicity (14%), metabolic disease (10%), autoimmune liver disease (6%), non-acetaminophen drug-related hepatotoxicity (5%), infections (6%), other diagnosed conditions (10%); 49% were indeterminate. Outcome varied between patient sub-groups; 20% with non-acetaminophen ALF died or underwent liver transplantation and never had clinical encephalopathy. CONCLUSIONS: Causes of ALF in children differ from in adults. Clinical encephalopathy may not be present in children. The high percentage of indeterminate cases provides an opportunity for investigation.