Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively

Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Fluorescence techniques for drug delivery research: theory and practice

Advances in drug delivery require an understanding of drug design, drug stability and metabolism together with the complexities imposed by the biological system such as cell/tissue penetration, drug-target interaction, and the pharmacodynamic consequences. Fluorescence microscopy provides a comprehensive tool for investigating many of these aspects of drug delivery in single cells and whole tissue. This review presents the fundamental concepts of fluorescence-based methodologies. The core principles which underlie the fluorescence process and the interpretation of these events drives instrument design and the components required to illuminate and detect fluorescent probes. Many drugs are inherently auto-fluorescent and therefore can be tracked using microscopy techniques, while other more indirect approaches assay the consequences of drug perturbation. This review addresses the two principle aims in fluorescence microscopy; to generate and enhance fluorescence-derived contrast that may reveal a central process of drug delivery

Gel formulations and uses thereof

The present invention provides the use of a composition comprising a block polymer as a support matrix in the manipulation, processing or analysis of particles, such as cells and fluorescent beads. In a preferred embodiment, the composition exhibits gel-sol thermoreversibility, micelle formation under gelling conditions, optically compatible, controllable surfactant properties, molecular sieving properties and biocompatibility. Further aspects of the invention provide (a) a support matrix composition comprising a block polymer, fluorescent beads and/or a dye for use in the manipulation, processing or analysis of particles, (b) a multichamber plate coated in a support matrix composition and (c) kits for producing the same

Cytomics and cellular informatics - coping with asymmetry and heterogeneity in biological systems

Cytomics has a practical role to play in drug discovery within the immediate limitations of cell-based analyses. A more elusive goal is the clarification of the complexity of a dynamic cellular response to a drug through mathematical modelling. The common aim for drug discovery and cytomics is to bridge the molecular–cellularsystems gap. The strategic challenges faced include: suitable preclinical biological models, cytometric platforms that resolve cellular or behavioural identity, analysis and visualization tools that act as integrating principles, consensus on standards and, finally, the implementation of informatics to serve the demands of decision making. Advances in all these key areas will help to address the need to identify and evaluate the origins of asymmetry and heterogeneity in cellularsystems and thus reveal new opportunities for therapeutic targeting

Microtrench and tumour proliferation assay

There is provided a cell culture microtrench being defined on or in a surface of a substrate, wherein the ratio of the width of the microtrench to the maximum length of the short axis of a cell type of interest is about 6 or preferably less, the length of the short axis of the cell type being measured when a cell is in detached or suspended form. There is also provided an array comprising such a microtrench and uses of such microtrenches, including cell-based assays