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Objective Asthma has a substantial impact on quality of life and health care resources. The identification of a more cost-effective, yet equally efficacious, treatment could positively influence the economic burden of this disease. Fluticasone propionate/Formoterol (FP/FOR) may be as effective as Fluticasone Salmeterol (FP/SAL). We evaluated non-inferiority of asthma control in terms of the proportion of patients free from exacerbations, and conducted a cost impact analysis. Methods This historical, matched cohort database study evaluated two treatment groups in the Optimum Patient Care Research Database in the UK: 1) an FP/FOR cohort of patients initiating treatment with FP/FOR or changing from FP/SAL to FP/FOR and; 2) an FP/SAL cohort comprising patients initiating, or remaining on FP/SAL pMDI combination therapy. The main outcome evaluated non-inferiority of effectiveness (defined as prevention of severe exacerbations, lower limit of the 95% confidence interval (CI) of the mean difference between groups in patient proportions with no exacerbations is −3.5% or higher) in patients treated with FP/FOR versus FP/SAL. Results After matching 1:3, we studied a total of 2472 patients: 618 in the FP/FOR cohort (174 patients initiated on FP/FOR and 444 patients changed to FP/FOR) and 1854 in the FP/SAL cohort (522 patients initiated FP/SAL and 1332 continued FP/SAL). The percentage of patients prescribed FP/FOR met non-inferiority as the adjusted mean difference in proportion of no severe exacerbations (95%CI) was 0.008 (−0.032, 0.047) between the two cohorts. No other significant differences were observed except acute respiratory event rates, which were lower for patients prescribed FP/FOR (rate ratio [RR] 0.82, 95% CI 0.71, 0.94). Conclusions Changing to, or initiating FP/FOR combination therapy, is associated with a non-inferior proportion of patients who are severe exacerbation-free at a lower average annual cost compared with continuing or initiating treatment with FP/SAL.

BACKGROUND: The lack of centralised data on severe asthma has resulted in a scarcity of information about the disease and its management. The development of a common data collection tool for the International Severe Asthma Registry (ISAR) will enable standardised data collection, subsequently enabling data interoperability.OBJECTIVES: To create a standardised list of variables for the first international registry for severe asthma via expert consensus.METHODS: A modified Delphi process was used to reach consensus on a minimum set of variables to capture in ISAR: the core variables. The Delphi panel brought together 27 international experts in the field of severe asthma research. The process consisted of three iterative rounds. In each round, all Delphi panel members were issued an electronic ISAR Delphi workbook to complete and return to the ISAR Delphi administrator. Workbooks and result summaries were anonymously distributed by the Delphi administrator to all panel members at subsequent rounds. Finalisation of the core variable list was facilitated by two face-to-face meetings.RESULTS: Of the initial 747 selected variables, the Delphi panel reached a consensus on 95. The chosen variables will allow severe asthma to be assessed against patient demographics and medical history, patient-reported outcomes, diagnostic information and clinical characteristics. Physician-reported outcomes such as non-adherence and information about treatment and management strategies will also be recorded.CONCLUSION: This is the first global attempt to generate an international severe asthma registry using a common set of core variables to ensure that data collected across all participating countries are standardised.

Chronic non-specific respiratory symptoms are difficult to manage. This trial aimed to evaluate the association between baseline fractional exhaled nitric oxide (FeNO) and the response to inhaled corticosteroids in patients with non-specific respiratory symptoms.In this double-blind randomised placebo-controlled trial, we enrolled undiagnosed patients, aged 18-80 years, with cough, wheeze, or dyspnoea and less than 20% bronchodilator reversibility across 26 primary care centres and hospitals in the UK and Singapore. Patients were assessed for 2 weeks before being randomly assigned (1:1) to 4 weeks of treatment with extrafine inhaled corticosteroids (QVAR 80 μg, two puffs twice per day, equivalent to 800 μg per day beclomethasone dipropionate) or placebo. Randomisation was stratified by baseline FeNO measurement: normal (≤25 parts per billion [ppb]), intermediate (25 tp40 ppb), and high (≥40 ppb). The primary endpoint was change in Asthma Control Questionnaire (ACQ7) mean score. We used generalised linear modelling to assess FeNO as a predictor of response, estimating an interaction effect between FeNO and treatment on change in ACQ7. We did our primary and secondary analyses in the per-protocol set, which excluded patients with non-completion of the primary endpoint, non-compliance to treatment (ascertained by patient report), and study visits made outside the predefined visit windows. This study is registered on ClinicalTrials.gov, number NCT02294279.Between Feb 4, 2015, and July 12, 2016, we randomly assigned 294 patients to extrafine inhaled corticosteroid treatment (n=148) or placebo (n=146). Following exclusions due to protocol violations, we analysed 214 patients (114 extrafine inhaled corticosteroids and 100 placebo). We observed a significant interaction between baseline FeNO and treatment group for every 10 ppb increase in baseline FeNO, with the change in ACQ7 greater in the extrafine inhaled corticosteroids group than in the placebo group (difference between groups 0·071, 95% CI 0·002 to 0·139; p=0·044). The most common adverse events were nasopharyngitis (18 [12%] patients in the treatment group vs 13 [9%] in the placebo group), infections and infestations (25 [17%] vs 21 [14%]), and respiratory, thoracic, and mediastinal disorders (13 [9%] vs 17 [12%]).FeNO measurement is an easy and non-invasive tool to use in clinical practice in patients with non-specific respiratory symptoms to predict response to inhaled corticosteroids. Further research is needed to examine its role in patients with evidence of other airway diseases, such as chronic obstructive pulmonary disease.Sponsored by OPRI with partial funding by Circassia and study drugs provided by TEVA.

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells.

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. MOE (Min. of Education, S’pore) Published version

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as "multitelomeric signals". Here, we report that TRF1 deficiency also leads to the formation of "ultra-fine bridges" (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.

Summary The TPP1/ACD protein (hereafter TPP1) is a component of the shelterin complex at mammalian telomeres. Here we find that Tpp1 -deficient mouse embryonic fibroblasts (MEFs) show increased chromosomal instability including sister chromatid fusions and chromosomes with multitelomeric signals related to telomere fragility. Tpp1 deletion decreases both TERT (the telomerase catalytic subunit) binding to telomeres in MEFs and telomerase function at chromosome ends in vivo. Abrogation of Tpp1 abolished net telomere elongation in the context of nuclear reprogramming of MEFs into induced pluripotent stem cells, whereas Tpp1 deletion in stratified epithelia of Tpp1 Δ/Δ K5-Cre mice resulted in perinatal death, severe skin hyperpigmentation, and impaired hair follicle morphogenesis. p53 deficiency rescues skin hyperpigmentation and hair growth in these mice, indicating that p53 restricts proliferation of Tpp1 -deficient cells. These results suggest a telomere-capping model where TPP1 protects telomere integrity and regulates telomerase recruitment to telomeres, thereby preventing early occurrence of degenerative pathologies.

Telomeres are transcribed from the telomeric C-rich strand, giving rise to UUAGGG repeat-containing telomeric transcripts or TERRA, which are novel structural components of telomeres. TERRA abundance is highly dependent on developmental status (including nuclear reprogramming), telomere length, cellular stresses, tumour stage and chromatin structure. However, the molecular mechanisms and factors controlling TERRA levels are still largely unknown. In this study, we identify a set of RNA-binding proteins, which endogenously bind and regulate TERRA in the context of primary mouse embryonic fibroblasts. The identification was carried out by biotin pull-down assays followed by LC-MALDI TOF/TOF mass spectrometry. Different members of the heterogeneous nuclear ribonucleoprotein family are among the ribonucleoprotein family that bind more abundantly to TERRA. Downregulation of TERRA-bound RBPs by small interfering RNA further shows that they can impact on TERRA abundance, their location and telomere lengthening. These findings anticipate an impact of TERRA-associated RBPs on telomere biology and telomeres diseases, such as cancer and aging.