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.