Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma

Background

Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.

Methods

In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.

Results

Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.

Conclusions

We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.     

A Brain-Computer Interface Based Cognitive Training System for Healthy Elderly: A Randomized Control Pilot Study for Usability and Preliminary Efficacy

Cognitive decline in aging is a pressing issue associated with significant healthcare costs and deterioration in quality of life. Previously, we reported the successful use of a novel brain-computer interface (BCI) training system in improving symptoms of attention deficit hyperactivity disorder. Here, we examine the feasibility of the BCI system with a new game that incorporates memory training in improving memory and attention in a pilot sample of healthy elderly. This study investigates the safety, usability and acceptability of our BCI system to elderly, and obtains an efficacy estimate to warrant a phase III trial. Thirty-one healthy elderly were randomized into intervention (n = 15) and waitlist control arms (n = 16). Intervention consisted of an 8-week training comprising 24 half-hour sessions. A usability and acceptability questionnaire was administered at the end of training. Safety was investigated by querying users about adverse events after every session. Efficacy of the system was measured by the change of total score from the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) before and after training. Feedback on the usability and acceptability questionnaire was positive. No adverse events were reported for all participants across all sessions. Though the median difference in the RBANS change scores between arms was not statistically significant, an effect size of 0.6SD was obtained, which reflects potential clinical utility according to Simon’s randomized phase II trial design. Pooled data from both arms also showed that the median change in total scores pre and post-training was statistically significant (Mdn = 4.0; p<0.001). Specifically, there were significant improvements in immediate memory (p = 0.038), visuospatial/constructional (p = 0.014), attention (p = 0.039), and delayed memory (p<0.001) scores. Our BCI-based system shows promise in improving memory and attention in healthy elderly, and appears to be safe, user-friendly and acceptable to senior users. Given the efficacy signal, a phase III trial is warranted.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01661894","term_id":"NCT01661894"}}NCT01661894     

The study of genetic diversity patterns of Coffea commersoniana, an endangered coffee species from Madagascar: a model for conservation of other littoral forest species

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.     

Comparison of HER2 and Phospho-HER2 Expression between Biopsy and Resected Breast Cancer Specimens Using a Quantitative Assessment Method

Background

HER2/Neu (ErbB-2) overexpression, which occurs in 15–20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2.

Methods

A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr¹²⁴⁸HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair.

Results

Both HER2 immunoreactivity (P<0.0001) and pTyr¹²⁴⁸HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr¹²⁴⁸HER2 showed no direct correlation with HER2 in either CNB or resection specimens.

Conclusions

The data suggest that measurement of both HER2 and phospho- Tyr¹²⁴⁸HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr¹²⁴⁸HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.     

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.     

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.     

Three Dimensional Structure of Mammalian Tachykinin Peptide Neurokinin B Bound to Lipid Micelles

Abstract

Neurokinin B (NKB), a decapeptide of mammalian origin exhibits a variety of biological activities such as regulatory functions in reproduction, pre-eclampsia and neuroprotection in Alzheimer's disease. In order to gain insight into structure-function relationship, three- dimensional structure of NKB has been investigated using CD spectropolarimetry and two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy in aqueous and membrane mimetic solvents. Unambiguous NMR assignments of resonances have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments. Distance constraints obtained from the NMR data have been used to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Our data show that a helical structure is induced in NKB, in presence of perdeuterated dodecyl phosphocholine (DPC) micelles, a membrane model system. Further, the conformation adopted by NKB in presence of DPC micelles represents a structural motif typical of neurokinin-3 selective agonists.