Understanding the lives of separating and separated families in the UK: what evidence do we need?

This study was designed to address three broad questions: What are the evidence – and data – needs around family separation in the UK? How far are these needs met by administrative, survey and other research data that currently exist or are in the process of being developed? What additional data are required, and how would these best be collected

Optimistic Planning for Markov Decision Processes

International audienceThe reinforcement learning community has recently intensified its interest in online planning methods, due to their relative independence on the state space size. However, tight near-optimality guarantees are not yet available for the general case of stochastic Markov decision processes and closed-loop, state-dependent planning policies. We therefore consider an algorithm related to AO* that optimistically explores a tree representation of the space of closed-loop policies, and we analyze the near-optimality of the action it returns after n tree node expansions. While this optimistic planning requires a finite number of actions and possible next states for each transition, its asymptotic performance does not depend directly on these numbers, but only on the subset of nodes that significantly impact near-optimal policies. We characterize this set by introducing a novel measure of problem complexity, called the near-optimality exponent. Specializing the exponent and performance bound for some interesting classes of MDPs illustrates the algorithm works better when there are fewer near-optimal policies and less uniform transition probabilities

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes

Generation of a genome scale lentiviral vector library for EF1α promoter-driven expression of hman ORFs and identification of human genes affecting viral titer

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY (R) cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors

Diagram summarising the key library construction steps.

<p>1. Gateway cloning was used to transfer ORFs into the lentiviral expression plasmid plv411G downstream of the EF1α promoter and upstream of the IRES driven GFP; 2. Virus production was performed by transfecting expression clones together with viral packaging plasmids into HEK293T cell line; 3. Viral supernatant was collected into a fresh set of plates and stored; 4. Transfected cells remaining in the plates were fixed and scanned to determine GFP fluorescence in each well. Frequency distribution histogram illustrates number of wells (y-axis) that had similar GFP fluorescence intensity (x-axis). Values between plates were normalized by zeroing on the mean of 4 mock transfected wells on each plate. Each plate also contained 4 empty expression vector wells, shown in green, which were used as positive controls; 5. Thirty eight ORF-expressing wells were randomly selected from each of the tail ends of the frequency distribution categories, to evaluate the performance of the viral supernatants on a range of target cell lines.</p

Library clone insert size and GFP fluorescence in virus-making cells.

<p>A: scatter-plot of cloned insert size and total well GFP fluorescence in transfected HEK293T cells. B: frequency distribution histogram of clones in different insert size category.</p

Effect of low virus titer-producing genes on viability of the transfected cells.

<p>HEK293T cells were transfected with gene (x-axis) expressing plasmids either alone or with viral packaging vector day after seeding. 21 h after transfection, medium was replaced with PBS containing Hoechst33342 and propidium iodide (PI), which stain DNA and dying cells respectively. Cells were scanned in blue (Hoechst, A) red (PI, B) and green (GFP, C) channels and number of objects determined in each channel. A and B represent independent object counts, data in C are expressed as proportion of GFP positive cells in the Hoechst stained population. A - Significantly reduced number of surviving cells in the well compared to untreated wells (CELLS): lipofectamine alone (MOCK) (P = 0.034); MOCK with packaging plasmids (P = 7.5E−10); all others (P≤1.1E−05) B - Significantly increased PI positive cells compared to vector: expression plasmid only BCL2L1 (P = 6.88E−05), C3ORF1(P = 0.03), MTCH1(P = 4.66E−05), PNMAL2 (P = 0.01) P values determined using Aspin-Welch test; Bars = mean of 4 wells, error bars = SD, CELLS-untreated well, MOCK-wells where expression plasmid has been omitted. AWAT2, and vector were high virus titer producing in previous experiments.</p