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Core binding factor β (CBFβ), the essential coregulator of RUNX transcription factors, is one of the most frequently mutated genes in estrogen receptor-positive (ER + ) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumor suppressor role for CBFβ. However, the impact of missense mutations and the loss of CBFβ in ER + breast cancer cells have not been determined. Here we demonstrate that missense mutations in CBFβ accumulate near the Runt domain-binding region. These mutations inhibit the ability of CBFβ to form CBFβ-Runx-DNA complexes. We further show that deletion of CBFβ, using CRISPR-Cas9, in ER + MCF7 cells results in an increase in cell migration. This increase in migration is dependent on the presence of ERα. Analysis of the potential mechanism revealed that the increase in migration is driven by the coregulation of Trefoil factor 1 (TFF1) by CBFβ and ERα. RUNX1-CBFβ acts to repress ERα-activated expression of TFF1. TFF1 is a motogen that stimulates migration and we show that knockdown of TFF1 in CBFβ -/- cells inhibits the migratory phenotype. Our findings reveal a new mechanism by which RUNX1-CBFβ and ERα combine to regulate gene expression and a new role for RUNX1-CBFβ in the prevention of cell migration by suppressing the expression of the motogen TFF1. IMPLICATIONS: Mutations in CBFβ contribute to the development of breast cancer by inducing a metastatic phenotype that is dependent on ER., (©2019 American Association for Cancer Research.)

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: A novel, inhalation-driven, multidose dry powder inhaler (MDPI) that does not require coordination of actuation with inhalation has been developed., Objective: To evaluate the efficacy and safety of albuterol MDPI versus placebo MDPI after chronic dosing in children with asthma., Methods: This phase III, double-blind, parallel-group study included children with asthma (ages, 4-11 years) with forced expiratory volume in 1 second (FEV1) of 50-95% of predicted. After a 14-day run-in period wherein the patients continued their current asthma therapy and received single-blind placebo MDPI, they were randomized to albuterol MDPI 90 μg per inhalation, two inhalations four times daily (total daily dose, 720 μg), or placebo for 3 weeks. Pulmonary function was assessed on days 1 and 22. Efficacy and safety were evaluated by measuring the baseline-adjusted percent-predicted FEV1 (PPFEV1) area under the time curve over 6 hours (AUC0-6) after the dose and adverse events, respectively., Results: The full analysis set included 184 patients. Patients treated with albuterol MDPI versus patients treated with placebo MDPI had significantly greater baseline-adjusted PPFEV1 AUC0-6 over 3 weeks (least squares mean difference, 25.0%•hour, which favored albuterol; p < 0.001). The benefit of albuterol (mean change in PPFEV1) was evident 5 minutes after dosing and lasted several hours; the maximal effect was noted 1 to 2 hours after dosing. Albuterol MDPI was well tolerated., Conclusions: In children with persistent asthma, albuterol MDPI improved pulmonary function significantly better than placebo MDPI over 3 weeks of treatment. Clinical efficacy was evident within 5 minutes of dosing and maintained for >2 hours. Four times daily administration was well tolerated.

All measurements performed in science and industry are based on the International System of Units, the SI. It has been proposed to revise the SI following an approach which was implemented for the redefinition of the unit of length, the metre, namely to define the SI units by fixing the numerical values of so-called defining constants, including c , h , e , k and N A . We will discuss the reasoning behind the revision, which will likely be put into force in 2018. Precision engineering was crucial to achieve the required small measurement uncertainties and agreement of measurement results for the defining constants.

A full understanding of RUNX gene function in different epithelial lineages has been thwarted by the lethal phenotypes observed when constitutively knocking out these mammalian genes. However temporal expression of the Runx genes throughout the different phases of mammary gland development is indicative of a functional role in this tissue. A few studies have emerged describing how these genes impact on the fate of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential, with implications for the transformed state. The importance of the RUNX/CBFβ core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFβ appearing in a comprehensive gene list of predicted breast cancer driver mutations. Nonetheless, the evidence to date shows that the RUNX genes can have dualistic outputs with respect to promoting or constraining breast cancer phenotypes, and that this may be aligned to individual subtypes of the clinical disease. We take this opportunity to review the current literature on RUNX and CBFβ in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

Cytoskeletal filamin A (FLNA) is an important protein involved in multiple cellular processes. Previous studies have shown that FLNA can promote or inhibit cancer growth and development; however, the mechanisms underlying these events are not fully understood. Here we show that, in both 293T and SaOS2 cells, knockdown of FLNA significantly enhanced transcription of RNA polymerase (pol) III-transcribed genes except for a subset of tRNA genes. In contrast, re-expression of FLNA in an FLNA-deficient melanoma cell line (A7) repressed transcription of all pol III-transcribed genes, suggesting that FLNA inhibits pol III transcription in a cell type-specific manner. Chromatin immunoprecipitation assays revealed that the repression of pol III gene transcription by FLNA correlates with the decreased occupancy of the RNA pol III transcription machinery at promoters. Immunofluorescence microscopy and coimmunoprecipitation assays revealed that FLNA can associate with the RNA pol III transcription machinery through its actin-binding domain within nuclei. Mechanistic analysis revealed that FLNA suppresses pol III gene transcription by confining the recruitment of the RNA pol III transcription machinery at the promoters of the genes that are sensitive to the alteration of FLNA expression. These findings not only extend the understanding of FLNA function in cells but also provide novel insights into the mechanism by which FLNA represses cell proliferation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Many children struggle with albuterol hydrofluoroalkane (HFA) inhalers. Albuterol multidose dry powder inhaler (MDPI) may simplify rescue bronchodilator use in children., Objective: To demonstrate the comparability of albuterol MDPI and albuterol HFA in children with asthma., Methods: This phase II, multicenter, double-blind, double-dummy, single-dose, five-period, crossover study randomized patients (ages 4-11 years) with persistent asthma and prestudy forced expiratory volume in 1 second (FEV1) of 60-90% of predicted to 1 of 10 treatment sequences that contained albuterol MDPI (90 and 180 μg), albuterol HFA (90 and 180 μg), and placebo MDPI and placebo HFA. Efficacy was evaluated by measuring the area under the baseline-adjusted percent-predicted FEV1-time curve over 6 hours (PPFEV1 AUC0-6) after dosing. Safety was evaluated by adverse events., Results: The full analysis set included 61 patients. Albuterol MDPI and albuterol HFA significantly improved PPFEV1 AUC0-6 versus placebo (p ≤ 0.0107). Mean improvement (± standard error [SE]) in PPFEV1 AUC0-6 versus placebo with albuterol MDPI at 90 and 180 μg was similar (21.2 ± 4.87 [95% confidence interval {CI}, 11.60-30.81], and 22.6 ± 4.87 [95% CI, 13.00-32.20], %·hour, respectively). Mean improvement (± SE) with albuterol HFA 180 μg was significantly (p = 0.0226) greater versus albuterol HFA 90 μg (23.7 ± 4.85 [95% CI, 14.13-33.23], and 12.5 ± 4.85 [95% CI, 2.93-22.05], %·hour, respectively). All doses of albuterol were well tolerated., Conclusion: Albuterol MDPI 90 and 180 μg and albuterol HFA 180 μg provided similar and significant FEV1 improvements versus placebo; albuterol HFA 90 μg was significant versus placebo but seemed less effective based on absolute improvements in FEV1. ClinicalTrials.gov identifier: NCT01899144.

Background: Many children struggle with the use of albuterol hydrofluoroalkane (HFA) inhalers. Albuterol multidose dry powder inhaler (MDPI) may simplify rescue bronchodilator use in children., Objective: To compare the pharmacokinetics (PK), pharmacodynamics (PD), and tolerability of albuterol MDPI and albuterol HFA after a single inhaled dose in children with asthma., Methods: This single-center, open-label, two-period crossover study randomized children to albuterol MDPI or HFA 180 μg on two treatment days with a 4- to 14-day washout. Plasma albuterol concentrations were measured before the dose and up to 10 hours after the dose to determine the primary PK values of area under the plasma concentration-versus-time curve from time 0 to the last measurable concentration (AUC0-t), maximum observed concentration (Cmax), and AUC from time 0 extrapolated to infinity (AUC0-inf). Heart rate and blood pressure before the dose and after the dose were monitored for PD effects, and adverse events (AE) were monitored for overall safety., Results: Fifteen children, ages 6-11 years, were included (PK, n = 13 for time to Cmax and terminal half-life of elimination; n = 12 for AUC and Cmax due to incomplete data). AUC0-t (geometric mean ratio [GMR] 1.056 [90% confidence interval {CI}, 0.88-1.268]) and AUC0-inf (GMR 0.971 [90% CI, 0.821-1.147]) were comparable between treatments. Cmax was larger for albuterol MDPI versus HFA (GMR 1.340 [90% CI, 1.098-1.636]). PD parameters between the treatments were comparable. No deaths, serious AEs, treatment-emergent AEs, or withdrawals due to AEs were reported for either treatment., Conclusion: Albuterol MDPI and albuterol HFA had comparable PK and PD in children after a single 180-μg dose. ClinicalTrails.gov identifiers NCT01899144 and NCT02126839.

This paper provides a perspective on the development of ultra-precision technologies: What drove their evolution and what do they now promise for the future as we face the consequences of consumption of the Earth's finite resources? Improved application of measurement is introduced as a major enabler of mass production, and its resultant impact on wealth generation is considered. This paper identifies the ambitions of the defence, automotive and microelectronics sectors as important drivers of improved manufacturing accuracy capability and ever smaller feature creation. It then describes how science fields such as astronomy have presented significant precision engineering challenges, illustrating how these fields of science have achieved unprecedented levels of accuracy, sensitivity and sheer scale. Notwithstanding their importance to science understanding, many science-driven ultra-precision technologies became key enablers for wealth generation and other well-being issues. Specific ultra-precision machine tools important to major astronomy programmes are discussed, as well as the way in which subsequently evolved machine tools made at the beginning of the twenty-first century, now provide much wider benefits.

Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease.

The transcription factor Runx2 is essential for the expression of a number of bone-specific genes and is primarily considered a master regulator of bone development. Runx2 is also expressed in mammary epithelial cells, but its role in the mammary gland has not been established. Here we show that Runx2 forms a novel complex with the ubiquitous transcription factor Oct-1 to regulate the expression of the mammary gland-specific gene beta-casein. The Runx2/Oct-1 complex forms on a Runx/octamer element which is highly conserved in casein promoters. Chromatin immunoprecipitation, RNA interference, promoter mutagenesis, and transient expression analyses were used to demonstrate that the Runx2/Oct-1 complex contributes to the transcriptional regulation of the beta-casein gene. Analysis of the complex revealed autoinhibitory domains for DNA binding in both the N-terminal and the C-terminal regions of Runx2. Oct-1 stimulates the recruitment of Runx2 to the beta-casein promoter by interacting with the C-terminal region of Runx2, suggesting that Oct-1 stimulates Runx2 recruitment by relieving the autoinhibition of Runx2 DNA binding. These findings demonstrate that Runx2 collaborates with Oct-1 and contributes to the expression of a mammary gland-specific gene.