Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines.

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4.

Spiritual distress and integrity in palliative and non-palliative patients.

Twenty-two patients in a Midlands acute hospital Trust supplied recorded narratives of their experience of spiritual distress, their hopes for spiritual integrity, and any means that were proving helpful in moving from distress to integrity. The research subjects included both patients in palliative care and those undergoing various therapies. There was little difference between the responses of these two groups.

Optimization procedure for small interfering RNA transfection in a 384-well format.

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format.

Role of the cysteine protease cathepsin S in neuropathic hyperalgesia.

Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up-regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury.

Histone deacetylase activities are required for innate immune cell control of Th1 but not Th2 effector cell function.

Histone deacetylases (HDACs) play a critical role in regulating gene expression and key biological processes. However, how HDACs are involved in innate immunity is little understood. Here, in this first systematic investigation of the role of HDACs in immunity, we show that HDAC inhibition by a small-molecule HDAC inhibitor (HDACi), LAQ824, alters Toll-like receptor 4 (TLR4)-dependent activation and function of macrophages and dendritic cells (DCs).

A quantitative high-throughput endothelial cell migration assay.

By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.

Development of comprehensive functional genomic screens to identify novel mediators of osteoarthritis.

Objective: The aim of this study was to develop high-throughput assays for the analysis of major chondrocyte functions that are important in osteoarthritis (OA) pathogenesis and methods for high-level gene expression and analysis in primary human chondrocytes.

Methods: In the first approach, complementary DNA (cDNA) libraries were constructed from OA cartilage RNA and full-length clones were selected. These cDNAs were transferred into a retroviral vector using Gateway Technology.

A quantitative high-throughput endothelial cell migration assay.

By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.

Divergent pathways of gene expression are activated by the RAGE ligands S100b and AGE-BSA.

Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of proinflammatory responses. However, our previous work revealed that RAGE-binding AGEs free of endotoxin were incapable of inducing vascular cell adhesion molecule-1 (VCAM-1) or tumor necrosis factor-alpha (TNF-alpha) expression. Thus, the objective of this study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems.

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites.

Gene expression profiling of epothilone A-resistant cells.

In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and Taxol, and cells which exhibit low resistance to Taxol but high resistance to epothilone A.

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts.

Disruption of three acid proteases in Aspergillus niger--effects on protease spectrum, intracellular proteolysis, and degradation of target proteins.

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the delta pepA and the delta pepB disruptants, repectively.

Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger.

We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage lambda genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199, 283, 227 and 881 bp, interrupted by three introns of 53, 69 and 59 bp.

Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger.

Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression.

Cloning and characterization of the pepE gene of Aspergillus niger encoding a new aspartic protease and regulation of pepE and pepC.

We have cloned the pepE gene of Aspergillus niger, encoding an aspartic protease (PEPE), by screening a lambda genomic DNA library with a heterologous probe, the Neurospora crassa gene coding for a vacuolar proteinase. Sequencing of pepE genomic and cDNA clones revealed that the gene contains three introns, which are 91, 56 and 58-bp long. The deduced protein consists of 398 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme.

Cloning and characterization of the pepD gene of Aspergillus niger which codes for a subtilisin-like protease.

Serine proteases constitute an important group of extra- and intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe.

Heterologous protein secretion directed by a repressible acid phosphatase system of Aspergillus niger.

A new expression-secretion system of Aspergillus niger which directs the secretion of heterologous proteins is described. The promoter and signal peptide-encoding region of the phosphate-repressible aphA gene of A. niger, when fused to the coding region of the human interferon alpha 2 (hIFN alpha 2)-encoding gene (hIFN alpha 2), drives the expression of this gene and the secretion of the hIFN alpha 2 protein.

Dual X-ray absorptiometry: a comparison between fan beam and pencil beam scans.

The recent introduction of dual X-ray absorptiometry (DXA) systems with fan beam instead of conventional pencil beam scanning geometry represents a significant technical advance in bone densitometry. This report describes phantom and in-vivo studies of the effect of the change in beam configuration on DXA measurements. Fan beam and pencil beam measurements acquired on one of the new generation scanners, the Hologic QDR-2000, were compared with scans performed on an earlier pencil beam model, the Hologic QDR-1000.

Characterization of an Aspergillus nidulans genomic DNA fragment conferring phosphate-non-repressible acid-phosphatase activity.

A clone from an Aspergillus nidulans library was identified by its ability to confer enhanced staining for acid phosphatase (APase) activity upon phosphatase-deficient A. nidulans mutants. This APase activity is not repressed by high phosphate concentrations in the medium.

Cloning and characterisation of pepC, a gene encoding a serine protease from Aspergillus niger.

We have cloned a gene, pepC, encoding a serine proteinase, PEPC, from Aspergillus niger by screening a phage lambda genomic DNA library with a gene (PRB1) from Saccharomyces cerevisiae which codes for proteinase YscB. The nucleotide (nt) sequence of pepC revealed that the gene is composed of two exons of 369 nt and 1230 nt separated by a single 70-nt intron. The deduced protein of 533 amino acids (aa) has a putative signal sequence for transport into the endoplasmic reticulum.

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI.