Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl) quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 μM, in agreement with its DNMT3A inhibitory potency.
Background and Purpose The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). Experimental Approach Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. Key Results Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16A inh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs. Conclusions and Implications Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca 2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.
Angiotensin II (Ang II), part of the renin-angiotensin-aldosterone system (RAS), is a potent vasoconstrictor and has been recently approved for use by the US Food and Drug Administration in high-output shock. Though not a new drug, the recently published Angiotensin II for the Treatment of High Output Shock (ATHOS-3) trial, as well as a number of retrospective analyses have sparked renewed interest in the use of Ang II, which may have a role in treating refractory shock. We describe refractory shock, the unique mechanism of action of Ang II, RAS dysregulation in shock, and the evidence supporting the use of Ang II to restore blood pressure. Evidence suggests that Ang II may preferentially be of benefit in acute kidney injury and acute respiratory distress syndrome, where the RAS is known to be disrupted. Additionally, there may be a role for Ang II in cardiogenic shock, angiotensin converting enzyme inhibitor overdose, cardiac arrest, liver failure, and in settings of extracorporeal circulation.
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Aly-Khan A Lalani;
Wanling Xie;
David A Braun;
Marina Kaymakcalan;
Dominick Bossé;
John A Steinharter;
Dylan J Martini;
Ronit Simantov;
Xun Lin;
Xiao X Wei;
Bradley A McGregor;
Rana R McKay;
Lauren C Harshman;
Toni K Choueiri
BACKGROUND: Antibiotic use alters commensal gut microbiota, which is a key regulator of immune homeostasis. OBJECTIVE: To investigate the impact of antibiotic use on clinical outcomes in metastatic renal cell carcinoma (mRCC) patients treated with systemic agents. DESIGN, SETTING, AND PARTICIPANTS: We analyzed two cohorts: an institutional cohort (n=146) receiving programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-based immune checkpoint inhibitors (ICIs) and a trial-database cohort (n=4144) receiving interferon-α (n=510), mammalian target of rapamycin (mTOR) inhibitors (n=660), and vascular endothelial growth factor targeted therapies (VEGF-TT; n=2974) on phase II/III clinical trials. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS: The association of antibiotic use (defined as use from 8 wk before to 4 wk after the initiation of anticancer therapy) with progression-free survival (PFS) and overall survival (OS) was evaluated using Cox regression, adjusted for known prognostic factors including International Metastatic RCC Database Consortium risk factors. RESULTS AND LIMITATIONS: Most patients were male, had clear cell histology, and were at an intermediate risk. Overall, in the institutional cohort, objective response rate (ORR) was 30%, PFS was 7.2 mo, and 1-yr OS was 77%. Antibiotic users (n=31, 21%) had a lower ORR (12.9% vs 34.8%, p=0.026) and shorter PFS (adjusted hazard ratio [HR]=1.96, 95% confidence interval [CI] 1.20-3.20, p=0.007) than antibiotic nonusers. In the trial-database cohort, antibiotic use (n=709, 17%) adversely impacted OS in patients treated with interferon (HR=1.62, 95% CI 1.13-2.31, p=0.008) or with VEGF-TT and prior cytokines (HR=1.65, 95% CI 1.04-2.62, p=0.033), but not patients treated with mTOR inhibitors or VEGF-TT without prior cytokines. Limitations include retrospective design, and limited details regarding concomitant medications and antibiotic indication/duration. CONCLUSIONS: Antibiotic use appears to reduce the efficacy of immunotherapy-based regimens in mRCC. The modulation of gut microbiota may play an important role in optimizing outcomes of patients treated with ICIs. PATIENT SUMMARY: We evaluated metastatic renal cell carcinoma patients and found that those who were treated with immunotherapy had worse outcomes if they also received antibiotics at the start of treatment. This study highlights the importance of judicious antibiotic use.
Anoctamin 1 (ANO1)/TMEM16A is a Cl<sup>−</sup> channel activated by intracellular Ca<sup>2+</sup> mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca<sup>2+</sup>. Here, we demonstrate that two helices, “reference” and “Ca<sup>2+</sup> sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca<sup>2+</sup>-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca<sup>2+</sup>-dependent activation because neutralization of these charges change the Ca<sup>2+</sup> sensitivity. We now predict that the Ca<sup>2+</sup> sensor helix attaches to the reference helix in the resting state, and as intracellular Ca<sup>2+</sup> rises, Ca<sup>2+</sup> acts on the sensor helix, which repels it from the reference helix. This Ca<sup>2+</sup>-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.
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Danielle M. McCarthy;
Terry C. Davis;
Jennifer P. King;
Rebecca J. Mullen;
Stacy C. Bailey;
Marina Serper;
Kara L. Jacobson;
Ruth Parker;
Michael S. Wolf
Recent studies have linked patient misunderstanding of label instructions for as needed (PRN) medications to dosing errors. This study conducted a preliminary field test of patient-centered PRN label instructions. Patients participated in a hypothetical dosing experiment and were randomized to a patient-centered label (referred to as "Take-Wait-Stop") or standard label. Participants were asked to demonstrate dosing the medicine over 24 hours. Three types of independent dosing errors were measured: (a) taking more than two pills at one time, (b) exceeding the maximum daily dose, and (c) waiting fewer than 4 hours between doses. Generalized linear models were used to assess the association between label type, health literacy, and sociodemographic characteristics. Participants' mean age was 39.8 years, 62.1% were female, 43.7% were White, and 72.4% had adequate literacy. Of participants, 31.8% who were shown the standard label demonstrated taking in excess of 6 pills in 24 hours compared with only 14.0% of participants who were shown the Take-Wait-Stop label (p =.05). Overall, only 1 person demonstrated he would take more than 2 pills in a single dose. Of the standard label group, 20.5% demonstrated dosing intervals of fewer than 4 hours compared with 23.3% of the Take-Wait-Stop label group (p=.75). In a multivariate model, participants who were exposed to the standard label were 2.5 times more likely to exceed the recommended maximum daily dose (95% CI [1.05, 7.70], p=.03). The Take-Wait-Stop label was beneficial in preventing participants from exceeding the maximum dose in 24 hours, although it did not significantly reduce other dosing errors.
A sensitive and specific caspase-3 sensor, based on a single enhanced green fluorescent protein (EGFP) to avoid the cross-talk from the overlap of cyan fluorescent protein (CFP) excitation or yellow fluorescent protein (YFP) emission spectra as observed in most FRET probes, was reported. A ratiometric protease sensor was designed by grafting a caspase-3-specific cleavage linker at a sensitive loop location (Glu172) relative to the chromophore, taking advantage of the EGFP's high resistance to proteases. To obtain optimal signal change and kinetic properties, two helical sequences were used to extend the caspase-3 cleavage linker and to improve enzymatic accessibility in solution. The comparison between dual cleavage sequence (DEVD) and a single sequence in caspase-3 sensors was also conducted. Moreover, to achieve enzymatic specificity, a recognition sequence (VDEVDG) with preferable residues from P5 to P1 was inserted into EGFP to create another sensor denoted as EGFP-C3B.
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Andrea Sikora Newsome;
Brian Murray;
Susan E. Smith;
Todd Brothers;
Mohammad A. Al-Mamun;
Aaron M. Chase;
Sandra Rowe;
Mitchell S. Buckley;
David Murphy;
John W. Devlin
What gets measured, gets improved. —Robert Sharma
Every critically ill patient requires care by a critical care pharmacist (CCP) for best possible outcomes. Indeed, these highly trained professionals generate benefit through direct patient care (eg, pharmacist-driven protocols, medication monitoring, etc), participation on the intensive care unit (ICU) interprofessional team (eg, pharmacotherapy recommendations, team education, etc), and leadership in the development and implementation of quality improvement initiatives.1 However, clinical CCP services are not provided for all ICU patients, and CCP staffing models often vary substantially across ICUs in a given hospital and among ICUs in the United States.2-4 In this narrative review, we use a gap analysis approach to define current levels of clinical CCP services, identify barriers to reaching an optimal level of these services, and propose strategies focused on expanding clinical CCP services and justifying those that currently exist.
Ixazomib is an oral proteasome inhibitor, approved in USA, Canada, Australia and Europe in combination with lenalidomide and dexamethasone, for the treatment of patients with multiple myeloma who have received at least one prior therapy. We report a population pharmacokinetic model-based analysis for ixazomib that was pivotal in describing the clinical pharmacokinetics of ixazomib, to inform product labelling. Plasma concentration–time data were collected from 755 patients who received oral or intravenous ixazomib in once- or twice-weekly schedules in ten trials, including the global phase III TOURMALINE-MM1 study. Data were analysed using nonlinear mixed-effects modelling (NONMEM software version 7.2, ICON Development Solutions, Hanover, MD, USA). Ixazomib plasma concentrations from intravenous and oral studies were described by a three-compartment model with linear distribution and elimination kinetics, including first-order linear absorption with a lag time describing the oral dose data. Body surface area on the volume of the second peripheral compartment was the only covariate included in the final model. None of the additional covariates tested including body surface area (1.2–2.7 m 2 ), sex, age (23–91 years), race, mild/moderate renal impairment and mild hepatic impairment were found to impact systemic clearance, suggesting that no dose adjustment is required based on these covariates. The geometric mean terminal disposition phase half-life was 9.5 days, steady-state volume of distribution was 543 L and systemic clearance was 1.86 L/h. The absolute bioavailability of an oral dose was estimated to be 58%.