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Sequential and compartmentalized action of Rabs, SNAREs and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells, playing important roles in forming the permeability barrier, and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic E. coli receptor. While it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immuno-microscopy of normal and mutant mouse urothelia showed that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently, a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also showed that keratin 20 (K20), which formed a chicken-wire network 150-300 nm below the apical membrane and had hole sizes allowing FV passage, defined a subapical compartment containing FVs primed and strategically located for fusion. Finally, we showed that Rab8/11 and Rab27b function in the same pathway, that Rab27b-knockout leads to uroplakin and Slp2-a destabilization, and that Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.
© 2016 by The American Society for Cell Biology.

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Extending the Classification Approach for Comparing Two Active Treatment Arms to Binary and Time-to-Event Outcomes.

For regulatory purposes, in a trial comparing two active treatments, a hypothesis such as noninferiority or superiority must be prespecified even when there is little known about how they compare against each other or when the objective is simply to identify the best. In this paper, we extend an alternative classification methodology, the classification approach of Qu et al (Statistics in Medicine, 30:3488-3495), to compare two active treatments when outcomes are binary and time-to-event variables. This method based on estimation approach instead of hypothesis testing can be useful when little prior information is available on which treatment has better efficacy. The entire decision space is divided into eight distinct possible outcomes based on predefined lower and upper non-inferiority margins, and the conclusion will be drawn according to the location of the confidence interval for relative risk or hazard ratio (or its logarithm transformation). We demonstrate theoretically that this method controls the misclassification rate at the specified level. We also illustrate the method by simulations and using data from a Phase 3 first-line nonsmall cell lung cancer study.

Oral Debio1143 (AT406), an antagonist of inhibitor of apoptosis proteins, combined with daunorubicin and cytarabine in patients with poor-risk acute myeloid leukemia–results of a phase I dose-escalation study.

Treatment of acute myeloid leukemia (AML) remains difficult owing to the development of treatment resistance, which might be overcome through antagonists of inhibitors of apoptosis proteins (IAPs).
The present multicenter, open-label, dose-escalation study aimed to evaluate the tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of Debio1143 (formerly AT-406), a new IAP antagonist, when given along with a standard &quot;7 plus 3 regimen&quot; of daunorubicin and cytarabine to poor-risk patients with AML during the induction cycle. Consecutive patient cohorts received once-daily 100, 200, 300, or 400 mg of oral Debio1143 on treatment days 1 to 5. Blood samples were collected regularly until hematologic recovery or response was documented. Bone marrow samples were collected on days 0, 14, and 29 and PK and PD samples on days 1, 3, 5, 8, and 10 and 1, 2, and 8, respectively.
Of the 29 enrolled patients, 23 completed the study. The most common adverse events of any grade deemed related to treatment were nausea (31% of patients), diarrhea (14%), and febrile neutropenia (14%). Exposure exceeded dose proportionality, without accumulation over 5 days. Inhibition of cellular IAP1 was detectable in the CD34/CD117(+) cells and blasts. A total of 11 patients (38%) achieved complete remission, most in the 100-mg dose cohort. Of these, 6 (56%) developed a relapse within the study period. The patients with a response more frequently showed plasma increases of tumor necrosis factor-α and interleukin-8 after the first dose of Debio1143.
Debio1143 ≤ 400 mg/d showed good tolerability in combination with daunorubicin and cytarabine. Additional studies in subsets of patients with AML are warranted.
Copyright © 2015 Elsevier Inc. All rights reserved.

TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.

Neuropsychiatric disease is one of the most common manifestations of human systemic lupus erythematosus, but the mechanisms remain poorly understood. In human brain microvascular endothelial cells in vitro, TNF-like weak inducer of apoptosis (TWEAK) decreases tight junction ZO-1 expression and increases the permeability of monolayer cell cultures. Furthermore, knockout (KO) of the TWEAK receptor, Fn14, in the MRL/lpr lupus mouse strain markedly attenuates neuropsychiatric disease, as demonstrated by significant reductions in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of neuropsychiatric lupus (NPSLE). Evaluating brain sections of MRL/lpr Fn14WT and Fn14KO mice, we found that Fn14KO mice displayed significantly decreased cellular infiltrates in the choroid plexus. To evaluate the integrity of the blood brain barrier (BBB) in MRL/lpr mice, Western blot for fibronectin, qPCR for iNOS, and immunohistochemical staining for VCAM-1/ICAM-1 were performed. We found preserved BBB permeability in MRL/lpr Fn14KO mice, attributable to reduced brain expression of VCAM-1/ICAM-1 and iNOS. Additionally, administration of Fc-TWEAK intravenously directly increased the leakage of a tracer (dextran-FITC) into brain tissue. Furthermore, MRL/lpr Fn14KO mice displayed reduced antibody (IgG) and complement (C3, C6, and C4a) deposition in the brain. Finally, we found that MRL/lpr Fn14KO mice manifested reduced neuron degeneration and hippocampal gliosis. Our studies indicate that TWEAK/Fn14 interactions play an important role in the pathogenesis of NPSLE by increasing the accumulation of inflammatory cells in the choroid plexus, disrupting BBB integrity, and increasing neuronal damage, suggesting a novel target for therapy in this disease.
Copyright © 2015 Elsevier Ltd. All rights reserved.

A Screening Assay Cascade to Identify and Characterize Novel Selective Estrogen Receptor Downregulators (SERDs).

Here, we describe an approach to identify novel selective estrogen receptor downregulator (SERD) compounds with improved properties such as oral bioavailability and the potential of increased efficacy compared to currently marketed drug treatments. Previously, methodologies such as Western blotting and transient cell reporter assays have been used to identify and characterize SERD compounds, but such approaches can be limited due to low throughput and sensitivity, respectively. We have used an endogenous cell-imaging strategy that has both the throughput and sensitivity to support a large-scale hit-to-lead program to identify novel compounds. A screening cascade with a suite of assays has been developed to characterize compounds that modulate estrogen receptor α (ERα)-mediated signaling or downregulate ERα levels in cells. Initially, from a focused high-throughput screening, novel ERα binders were identified that could be modified chemically into ERα downregulators. Following this, cellular assays helped determine the mechanism of action of compounds to distinguish between on-target and off-target compounds and differentiate SERDs, selective estrogen receptor modulator (SERM) compounds, and agonist ERα ligands. Data are shown to exemplify the characterization of ERα-mediated signaling inhibitors using a selection of literature compounds and illustrate how this cascade has been used to drive the chemical design of novel SERD compounds.
© 2015 Society for Laboratory Automation and Screening.