Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment

In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.     

Psychophysiological responses to stress after stress management training in patients with rheumatoid arthritis

Background

Stress management interventions may prove useful in preventing the detrimental effects of stress on health. This study assessed the effects of a stress management intervention on the psychophysiological response to stress in patients with rheumatoid arthritis (RA).

Methods

Seventy-four patients with RA, who were randomly assigned to either a control group or a group that received short-term stress management training, performed a standardized psychosocial stress task (Trier Social Stress Test; TSST) 1 week after the stress management training and at a 9-week follow-up. Psychological and physical functioning, and the acute psychophysiological response to the stress test were assessed.

Results

Patients in the intervention group showed significantly lower psychological distress levels of anxiety after the training than did the controls. While there were no between-group differences in stress-induced tension levels, and autonomic (α-amylase) or endocrine (cortisol) responses to the stress test 1 week after the intervention, levels of stress-induced tension and cortisol were significantly lower in the intervention group at the 9-week follow-up. Overall, the response to the intervention was particularly evident in a subgroup of patients with a psychological risk profile.

Conclusion

A relatively short stress management intervention can improve psychological functioning and influences the psychophysiological response to stress in patients with RA, particularly those psychologically at risk. These findings might help understand how stress can affect health and the role of individual differences in stress responsiveness.

Trial Registration

TrialRegister.nl NTR1193     

Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation

The signaling events that regulate vascular tone include voltage-dependent Ca(2+) influx and the activities of various ionic channels; which molecular entities are involved and their role are still a matter of debate. Here we show expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in rat aortic smooth muscle cells. Immunoprecipitation and in vitro protein kinase A phosphorylation show the appearance of mature band C of CFTR. An immunohistochemistry study shows CFTR proteins in smooth muscles of aortic rings but not in skeletal muscles. Using the iodide efflux method, a combination of agonists and pharmacological agents was used to dissect the function of CFTR. Agonists of the cAMP pathway, the beta-adrenergic agonist isoproterenol, and the neuropeptide vasoactive intestinal peptide activate CFTR-dependent transport from cells maintained in a high but not low extracellular potassium-rich saline, suggesting that depolarization of smooth muscle is critical to CFTR activation. Smooth muscle CFTR possesses all of the pharmacological attributes of its epithelial homologues: stimulation by the CFTR pharmacological activators MPB-07 (EC(50) = 158 microm) and MPB-91 (EC(50) = 20 microm) and inhibition by glibenclamide and diphenylamine-2-carboxylic acid but not by 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene. Contraction measurements on isolated aortic rings were performed to study the contribution of CFTR to vascular tone. With aortic rings (without endothelium) preconstricted by high K(+) saline or by the alpha-adrenergic agonist norepinephrine, CFTR activators produced a concentration-dependent relaxation. These results identify for the first time the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone.     

Emergence of a drug-dependent human immunodeficiency virus type 1 variant during therapy with the T20 fusion inhibitor

The fusion inhibitor T20 belongs to a new class of anti-human immunodeficiency virus type 1 (HIV-1) drugs designed to block entry of the virus into the host cell. However, the success of T20 has met with the inevitable emergence of drug-resistant HIV-1 variants. We describe an evolutionary pathway taken by HIV-1 to escape from the selective pressure of T20 in a treated patient. Besides the appearance of T20-resistant variants, we report for the first time the emergence of drug-dependent viruses with mutations in both the HR1 and HR2 domains of envelope glycoprotein 41. We propose a mechanistic model for the dependence of HIV-1 entry on the T20 peptide. The T20-dependent mutant is more prone to undergo the conformational switch that results in the formation of the fusogenic six-helix bundle structure in gp41. A premature switch will generate nonfunctional envelope glycoproteins (dead spikes) on the surface of the virion, and T20 prevents this abortive event by acting as a safety pin that preserves an earlier prefusion conformation.     

Protein interaction verification and functional annotation by integrated analysis of genome-scale data

Assays capable of determining the properties of thousands of genes in parallel present challenges with regard to accurate data processing and functional annotation. Collections of microarray expression data are applied here to assess the quality of different high-throughput protein interaction data sets. Significant differences are found. Confidence in 973 out of 5342 putative two-hybrid interactions from S. cerevisiae is increased. Besides verification, integration of expression and interaction data is employed to provide functional annotation for over 300 previously uncharacterized genes. The robustness of these approaches is demonstrated by experiments that test the in silico predictions made. This study shows how integration improves the utility of different types of functional genomic data and how well this contributes to functional annotation.     

The knee adduction moment measured with an instrumented force shoe in patients with knee osteoarthritis

The external knee adduction moment (KAdM) during gait is an important parameter in patients with knee osteoarthritis (OA). KAdM measurement is currently restricted to instruments only available in gait laboratories. However, ambulatory movement analysis technology, including instrumented force shoes (IFS) and inertial and magnetic measurement systems (IMMS), can measure kinetics and kinematics of human gait free of laboratory restrictions. The objective of this study was a quantitative validation of the accuracy of the KAdM in patients with knee OA, when estimated with an ambulatory-based method (AmbBM) versus a laboratory-based method (LabBM). AmbBM is employing the IFS and a linked-segment model, while LabBM is based on a force plate and optoelectronic marker system. Effects of ground reaction force (GRF), centre of pressure (CoP), and knee joint position measurement are evaluated separately. Twenty patients with knee OA were measured. The GRFs showed differences up to 0.22 N/kg, the CoPs showed differences up to 4 mm, and the medio-lateral and vertical knee position showed differences to 9 mm, between AmbBM and LabBM. The GRF caused an under-estimation in KAdM in early stance. However, this effect was counteracted by differences in CoP and joint position, resulting in a net 5% over-estimation. In midstance and late stance the accuracy of the KAdM was mainly limited by use of the linked-segment model for joint position estimation, resulting in an under-estimation (midstance 6% and late stance 22%). Further improvements are needed in the estimation of joint position from segment orientation.     

Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.