Decoding pooled RNAi screens by means of barcode tiling arrays

<p>Abstract</p> <p>Background</p> <p>RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool.</p> <p>Results</p> <p>We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach.</p> <p>Conclusions</p> <p>In summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.</p

Optimising the Inflammatory Bowel Disease Unit to Improve Quality of Care:Expert Recommendations

INTRODUCTION: The best care setting for patients with inflammatory bowel disease [IBD] may be in a dedicated unit. Whereas not all gastroenterology units have the same resources to develop dedicated IBD facilities and services, there are steps that can be taken by any unit to optimise patients' access to interdisciplinary expert care. A series of pragmatic recommendations relating to IBD unit optimisation have been developed through discussion among a large panel of international experts.METHODS: Suggested recommendations were extracted through systematic search of published evidence and structured requests for expert opinion. Physicians [n = 238] identified as IBD specialists by publications or clinical focus on IBD were invited for discussion and recommendation modification [Barcelona, Spain; 2014]. Final recommendations were voted on by the group. Participants also completed an online survey to evaluate their own experience related to IBD units.RESULTS: A total of 60% of attendees completed the survey, with 15% self-classifying their centre as a dedicated IBD unit. Only half of respondents indicated that they had a defined IBD treatment algorithm in place. Key recommendations included the need to develop a multidisciplinary team covering specifically-defined specialist expertise in IBD, to instil processes that facilitate cross-functional communication and to invest in shared care models of IBD management.CONCLUSIONS: Optimising the setup of IBD units will require progressive leadership and willingness to challenge the status quo in order to provide better quality of care for our patients. IBD units are an important step towards harmonising care for IBD across Europe and for establishing standards for disease management programmes.</p

Unmet Medical Needs in Ulcerative Colitis: An Expert Group Consensus

BACKGROUND The authors aimed to conduct an extensive literature review and consensus meeting to identify unmet needs in ulcerative colitis (UC) and ways to overcome them. UC is a relapsing and remitting inflammatory bowel disease with varied, and changing, incidence rates worldwide. UC has an unpredictable disease course and is associated with a high health economic burden. During 2016 and 2017, a panel of experts was convened to identify, discuss and address areas of unmet need in UC. METHODS PubMed and Cochrane Library databases were searched for relevant articles describing studies performed in patients with UC. These findings were used to generate a set of statements relating to unmet needs in UC. Consensus on these statements was then sought from a panel of 9 expert gastroenterologists using a modified Delphi review process that consisted of anonymous surveys followed by live meetings. RESULTS In 2 literature reviews, over 5,000 unique records were identified and a total of 138 articles were fully reviewed. These were used to consider 26 areas of unmet need, which were explored in 2 face-to-face meetings, in which the statements were debated and amended, resulting in consensus on 30 final statements. The unmet needs identified were categorised into 7 areas: impact of UC on patients' daily life; importance of early diagnosis and treatment; drawbacks of existing treatments; urgent need for new treatments; and disease-, practice- or patient-focused unmet needs. CONCLUSIONS These expert group meetings found a number of areas of unmet needs in UC, which is an important first step in tackling them in the future. Future research and development should be focused in these areas for the management of patients with UC

Association Between Response to Etrolizumab and Expression of Integrin αE and Granzyme A in Colon Biopsies of Patients With Ulcerative Colitis

Background & AimsEtrolizumab is a humanized monoclonal antibody against the β7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did.MethodsWe performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1.ResultsColon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell−associated genes than patients who did not respond (P < .05). Colonic CD4+ integrin αE+ cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4+ αE− cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMAhigh vs 19% GZMAlow and 44% ITGAEhigh vs 19% ITGAElow). Compared with ITGAElow and GZMAlow patients, patients with ITGAEhigh and GZMAhigh had higher baseline numbers of epithelial crypt-associated integrin αE+ cells (P < .01 for both), but a smaller number of crypt-associated integrin αE+ cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%−80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline.ConclusionsLevels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarkerhigh patients. Larger, prospective studies of markers are needed to assess their clinical value

Consensus recommendations for patient-centered therapy in mild-to-moderate ulcerative colitis: the i Support Therapy–Access to Rapid Treatment (iSTART) approach

Symptomatic ulcerative colitis (UC) can be a chronic, disabling condition. Flares in disease activity are associated with many of the negative impacts of mild-to-moderate UC. Rapid resolution of flares can provide benefits to patients and healthcare systems. i Support Therapy–Access to Rapid Treatment (iSTART) introduces patient-centered care for mild-to-moderate UC. iSTART provides patients with the ability to self-assess symptomology and self-start a short course of second-line treatment when necessary. An international panel of experts produced consensus statements and recommendations. These were informed by evidence from systematic reviews on the epidemiology, mesalazine (5-ASA) treatment, and patient use criteria for second-line therapy in UC. Optimized 5-ASA is the first-line treatment in all clinical guidelines, but may not be sufficient to induce remission in all patients. Corticosteroids should be prescribed as second-line therapy when needed, with budesonide MMX® being a preferred steroid option. Active involvement of suitable patients in management of UC flares has the potential to improve therapy, with patients able to show good accuracy for flare self-assessment using validated tools. There is a place in the UC treatment pathway for an approach such as iSTART, which has the potential to provide patient, clinical and economic benefits

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis . Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene ( gelE ). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2 −/− ) mice were used to investigate the role of this receptor in E. faecalis -induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2 −/− mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2 −/− mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2 −/− mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2

ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα−, PR−, and Her2−, named accordingly “Triple-Negative” (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5–12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygroR) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50–100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships