Efficacy and Safety of Garadacimab in Combination with Standard of Care Treatment in Patients with Severe COVID-19.

BACKGROUND: Garadacimab, a fully human IgG4 monoclonal antibody, inhibits the kallikrein-kinin pathway at a key initiator, activated coagulation factor XII (FXIIa), and may play a protective role in preventing the progression of COVID-19. This phase 2 study evaluated the efficacy and safety of garadacimab plus standard of care (SOC) versus placebo plus SOC in patients with severe COVID-19. METHODS: Patients hospitalised with COVID-19 were randomised (1:1) to a single intravenous dose of garadacimab (700 mg) plus SOC or placebo plus SOC. Co-primary endpoint was incidence of endotracheal intubation or death between randomisation and Day 28. All-cause mortality, safety and pharmacokinetic/pharmacodynamic parameters were assessed. RESULTS: No difference in incidence of tracheal intubation or death (p = 0.274) or all-cause mortality was observed (p = 0.382). Garadacimab was associated with a lower incidence of treatment-emergent adverse events (60.3% vs 67.8%) and fewer serious adverse events (34 vs 45 events) versus placebo. No garadacimab-related deaths or bleeding events were reported, including in the 45.9% (n = 28/61) of patients who received concomitant heparin. Prolonged activated partial thromboplastin time (aPTT), and increased coagulation factor XII (FXII) levels were observed with garadacimab versus placebo to Day 14, whilst FXIIa-mediated kallikrein activity (FXIIa-mKA) was suppressed to Day 28. CONCLUSION: In patients with severe COVID-19, garadacimab did not confer a clinical benefit over placebo. Transient aPTT prolongation and suppressed FXIIa-mKA showed target engagement of garadacimab that was not associated with bleeding events even with concomitant anticoagulant use. The safety profile of garadacimab was consistent with previous studies in patients with hereditary angioedema. GOV IDENTIFIER: NCT04409509. Date of registration: 28 May, 2020.

The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling BRF1 and GTF3C2 expression in human cells.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

Prospective observational study of early respiratory management in preterm neonates less than 35 weeks of gestation.

BACKGROUND: Current guidelines for management of respiratory distress syndrome (RDS) recommend continuous positive airway pressure (CPAP) as the primary mode of respiratory support even in the most premature neonates, reserving endotracheal intubation (ETI) for rescue surfactant or respiratory failure. The incidence and timing of ETI in practice is poorly documented. METHODS: In 27 Level III NICUs in the US (n = 19), Canada (n = 3) and Poland (n = 5), demographics and baseline characteristics, respiratory support modalities including timing of ETI, administration of surfactant and caffeine/other methylxanthines, and neonatal morbidities were prospectively recorded in consecutive preterm neonates following written parental consent. Infants were divided into three groups according to gestational age (GA) at birth, namely 26-28, 29-32 and 33-34 weeks. Statistical comparisons between groups were done using Chi-Square tests. RESULTS: Of 2093 neonates (US = 1507, 254 Canada, 332 Poland), 378 (18%) were 26-28 weeks gestational age (GA), 835 (40%) were 29-32 weeks, and 880 (42%) were 33-34 weeks. Antenatal steroid use was 81% overall, and approximately 89% in neonates ≤32 weeks. RDS incidence and use of ventilatory or supplemental oxygen support were similar across all sites. CPAP was initiated in 43% of all infants, being highest in the 29-32-week group, with a lower proportion in other GA categories (p < 0.001). The overall rate of ETI was 74% for neonates 26-28 weeks (42% within 15 min of birth, 49% within 60 min, and 57% within 3 h), 33% for 29-32 weeks (13 16 and 21%, respectively), and 16% for 33-34 weeks (5, 6 and 8%, respectively). Overall intubation rates and timing were similar between countries in all GAs. Rates within each country varied widely, however. Across US sites, overall ETI rates in 26-28-week neonates were 30-60%, and ETI within 15 min varied from 0 to 83%. Similar within 15-min variability was seen at Polish sites (22-67%) in this GA, and within all countries for 29-32 and 33-34-week neonates. CONCLUSION: Despite published guidelines for management of RDS, rate and timing of ETI varies widely, apparently unrelated to severity of illness. The impact of this variability on outcome is unknown but provides opportunities for further approaches which can avoid the need for ETI.

A transcription factor IIA-binding site differentially regulates RNA polymerase II-mediated transcription in a promoter context-dependent manner.

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.

HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells.

BACKGROUND: Hypoxia stimulates metastasis in cancer and is linked to poor patient prognosis. In tumours, oxygen levels vary and hypoxic regions exist within a generally well-oxygenated tumour. However, whilst the heterogeneous environment is known to contribute to metastatic progression, little is known about the mechanism by which heterogeneic hypoxia contributes to cancer progression. This is largely because existing experimental models do not recapitulate the heterogeneous nature of hypoxia. The primary effector of the hypoxic response is the transcription factor Hypoxia inducible factor 1-alpha (HIF1-alpha). HIF1-alpha is stabilised in response to low oxygen levels in the cellular environment and its expression is seen in hypoxic regions throughout the tumour. METHODS: We have developed a model system in which HIF1-alpha can be induced within a sub-population of cancer cells, thus enabling us to mimic the effects of heterogeneic HIF1-alpha expression. RESULTS: We show that induction of HIF1-alpha not only recapitulates elements of the hypoxic response in the induced cells but also results in significant changes in proliferation, gene expression and mammosphere formation within the HIF1-alpha negative population. CONCLUSIONS: These findings suggest that the HIF1-alpha expressing cells found within hypoxic regions are likely to contribute to the subsequent progression of a tumour by modifying the behaviour of cells in the non-hypoxic regions of the local micro-environment.

Cytoskeletal Filamin A Differentially Modulates RNA Polymerase III Gene Transcription in Transformed Cell Lines.

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.

Runx Genes in Breast Cancer and the Mammary Lineage.

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/CBFß core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFß 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 CBFß in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

Albuterol multidose dry powder inhaler efficacy and safety versus placebo in children with asthma.

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.

Contributions of precision engineering to the revision of the SI.

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.

Pharmacokinetics and pharmacodynamics of albuterol multidose dry powder inhaler and albuterol hydrofluoroalkane in children with asthma.

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.

Albuterol multidose dry powder inhaler and albuterol hydrofluoroalkane versus placebo in children with persistent asthma.

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.

Acetazolamide therapy for metabolic alkalosis in critically ill pediatric patients.

OBJECTIVE: Despite a paucity of supporting literature, acetazolamide is commonly used in critically ill children with metabolic alkalosis (elevated plasma bicarbonate [pHco-3] and pH). The objective of this study was to assess the change in 18 hours after initiation of acetazolamide therapy. DESIGN: Retrospective study. SETTING: PICU of an urban, tertiary-care children's hospital. PATIENTS: Mechanically ventilated children (≤ 17 yr) with metabolic alkalosis (pHco-3 ≥ 35 mmol/L). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 153 consecutively screened patients, 61 patients (29 female patients) were enrolled: 18 cardiac patients (after congenital heart disease repair) and 43 noncardiac patients. The cardiac patients were younger than the noncardiac patients (median [interquartile range] age, 0.6 mo [0.3-2.5 mo] vs 7.4 mo [2.8-39.9 mo]; p < 0.00001) and had higher preacetazolamide baseline diuretic scores and urine output. The pHco-3 levels 18 hours after initiation of acetazolamide were reduced in the cohort as a whole (40.2 ± 4.8 to 36.2 ± 5.6 mmol/L; p < 0.001) and in the noncardiac patients, but they were unchanged in the cardiac patients. The PCO2 remained unchanged after acetazolamide in both subgroups. Because young age and presence of cardiac disease were potential confounders, the 20 noncardiac patients who are 6 months old or younger were compared with the cardiac subgroup and demonstrated reduced pHco-3 after acetazolamide and lower preacetazolamide baseline diuretic score and urine output. CONCLUSION: Acetazolamide reduces pHco-3 concentration in critically ill, mechanically ventilated children overall, but it did not do so in cardiac patients in our cohort, even in comparison with noncardiac patients of a similar age. These findings do not support the current use of acetazolamide for metabolic alkalosis in critically ill children with congenital heart disease. Further study is required to determine why these cardiac patients respond differently to acetazolamide than noncardiac patients and whether this response impacts important clinical outcomes, for example, weaning mechanical ventilation.

Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

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.

Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch.

The epidermis is a highly organized structure, the integrity of which is central to the protection of an organism. Development and subsequent maintenance of this tissue depends critically on the intricate balance between proliferation and differentiation of a resident stem cell population; however, the signals controlling the proliferation-differentiation switch in vivo remain elusive. Here, we show that mice carrying a homozygous missense mutation in interferon regulatory factor 6 (Irf6), the homolog of the gene mutated in the human congenital disorders Van der Woude syndrome and popliteal pterygium syndrome, have a hyperproliferative epidermis that fails to undergo terminal differentiation, resulting in soft tissue fusions. We further demonstrate that mice that are compound heterozygotes for mutations in Irf6 and the gene encoding the cell cycle regulator protein stratifin (Sfn; also known as 14-3-3sigma) show similar defects of keratinizing epithelia. Our results indicate that Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch and that Irf6 and Sfn interact genetically in this process.

An under-recognized benefit of cardiopulmonary resuscitation: organ transplantation.

OBJECTIVE: For many patients who suffer cardiac arrest, cardiopulmonary resuscitation does not result in long-term survival. For some of these patients, the evolution to donation of organs becomes an option. Organ transplantation after cardiopulmonary resuscitation is not reported as an outcome of cardiopulmonary resuscitation and is therefore overlooked. We sought to determine the number and proportion of organs transplanted from donors who received cardiopulmonary resuscitation after a cardiac arrest in the United States and to compare survival of organs from donors who had cardiopulmonary resuscitation (cardiopulmonary resuscitation organs) versus donors who did not have resuscitation (noncardiopulmonary resuscitation organs). DATA SOURCE: We retrospectively analyzed a nationwide, population-based database of all organ donors and recipients from the United Network for Organ Sharing between July 1999 and June 2011. STUDY SELECTION: We queried the database for all organs from deceased donors between July 1999 and June 2011. Organs from living donors (n = 76,015), all organs with missing cardiopulmonary resuscitation data (n = 59), and organs procured following a circulatory determination of death (n = 12,030) were excluded. DATA EXTRACTION: We report donor demographic data and organ survival outcomes among organs from donors who received cardiopulmonary resuscitation (cardiopulmonary resuscitation organs) and donors who had not received cardiopulmonary resuscitation (noncardiopulmonary resuscitation organs). Graft survival of cardiopulmonary resuscitation organs versus noncardiopulmonary resuscitation organs was compared using Kaplan-Meier estimates and stratified log-rank test. DATA SYNTHESIS: In the United States, among the 224,076 organs donated by donors who were declared dead by neurologic criteria between 1999 and 2011, at least 12,351 organs (5.5%) were recovered from donors who received cardiopulmonary resuscitation. Graft survival of cardiopulmonary resuscitation organs was not significantly different than that of noncardiopulmonary resuscitation organs. CONCLUSIONS: At least 1,000 organs transplanted per year in the United States (> 5% of all organs transplanted from patients declared dead by neurologic criteria) are recovered from patients who received cardiopulmonary resuscitation. Organ recovery and successful transplantation is an unreported beneficial outcome of cardiopulmonary resuscitation.

Development of a bedside tool to predict time to death after withdrawal of life-sustaining therapies in infants and children.

OBJECTIVES: To generate a preliminary bedside predictor of rapid time-to-death after withdrawal of support in children to help identify potential candidates for organ donation after circulatory death. DESIGN: Retrospective chart review. SETTING: Pediatric intensive care unit of an academic children's hospital. PATIENTS: All deaths in the pediatric intensive care unit from May 1996 to April 2007. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among 1389 deaths, 634 patients underwent withdrawal of support and 518 with complete data regarding demographics, life-supportive therapies, and end-of-life circumstances were analyzed. Three hundred seventy-three (72%) patients died within 30 mins of withdrawal and 452 (87%) died within 60 mins. Using multiple logistic regression, significant predictors of death within 30 or 60 mins (typical cut-off times for organ donation) were identified and a predictor score was generated. Significant predictors included: age 1 month or younger; norepinephrine, epinephrine, or phenylephrine >0.2 µg/kg/min; extracorporeal membrane oxygenation; and positive end-expiratory pressure >10 cmH2O; and spontaneous ventilation. Possible scores for the 30-min predictor ranged from -17 to 67; a score ≤-9 predicted a 37% probability of death ≤ 30 mins, whereas a score ≥ 38 predicted an 85% probability of death within 30 mins. For the 60-min predictor, scores ranged from -21 to 38; score ≤-10 predicted a 59% probability of death within 60 mins and a score ≥ 16 predicted a 98% probability of death within 60 mins. CONCLUSIONS: This tool is a reasonable preliminary predictor for death within 30 or 60 mins after withdrawal of support in terminally ill or injured children and might assist in identifying potential pediatric candidates for donation after circulatory death, although prospective validation is required.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFß-dependent expression of the Wnt antagonist, sclerostin.

INTRODUCTION: Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown. METHODS: The aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFß) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFß. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFß-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells. RESULTS: We show that Runx2/CBFß mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFß. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFß in metastatic breast cancer cells. CONCLUSIONS: This study demonstrates that Runx2 and CBFß are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.

Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6.

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype-phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype-phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

The Runx transcriptional co-activator, CBFbeta, is essential for invasion of breast cancer cells.

BACKGROUND: The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFbeta is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFbeta is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFbeta-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFbeta. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFbeta expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFbeta. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. RESULTS: We show that CBFbeta is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFbeta contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFbeta is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFbeta is recruited to both the Osteopontin and the Galectin-3 promoters. CONCLUSIONS: CBFbeta is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFbeta is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFbeta is essential for invasion, not all Runx2-target genes require CBFbeta. We conclude that CBFbeta is required for a subset of Runx2-target genes that are sufficient to maintain the invasive phenotype of the cells. These findings suggest that the interaction between Runx2 and CBFbeta might represent a viable target for therapeutic intervention to inhibit bone metastasis.