The potassium channel interacting protein 3 (DREAM/KChIP3) heterodimerizes with and regulates calmodulin function.

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 µM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

The regulation of apoptosis by the downstream regulatory element antagonist modulator/potassium channel interacting protein 3 (DREAM/KChIP3) through interactions with hexokinase I.

The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.

The short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes.

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.

Expression and characterization of the soluble form of recombinant mature HSV-2 glycoprotein G for use in anti-HSV-2 IgG serodiagnostic immunoassay.

Herpes simplex virus type-2 (HSV-2) specific glycoprotein G (gG-2) is widely used as the antigen of choice for serodiagnosis of HSV-2. In order to develop an ELISA for serodetection of HSV-2 IgG in patient sera, the soluble form of the mature gG-2 antigen (mgG-2), gG283-649, was expressed using a baculovirus expression system. gG283-649 contains the complete extracellular domain of mgG-2 including the C-terminal region, which despite homology to gG-1, does not cross-react with HSV-1 antibodies present in HSV-1 positive patient sera. gG283-649 had increased performance compared to a previously described gG-2 fragment and showed high sensitivity and specificity in a method comparison with HerpeSelect 1 & 2 Immunoblot IgG, a commercially available FDA-cleared assay for serodetection of HSV-1 and 2 antibodies. A total of 234 clinical samples consisting of 134 high risk samples, including 45 samples from pregnant subjects, and a panel of 100 mixed diagnosis samples, spanning the measurable range were tested in the method comparison. Clinical sensitivity and specificity were determined to be 94.2% and 100%, respectively. We conclude that this soluble form of mgG-2 is a novel antigen of choice for developing an ELISA for type-specific serodiagnosis of HSV-2.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

Building Community Through a #pulmcc Twitter Chat to Advocate for Pulmonary, Critical Care, and Sleep.

BACKGROUND: Social media sites such as Twitter can significantly enhance education and advocacy efforts. In 2013, the American College of Chest Physicians (CHEST) launched a Twitter chat series using the hashtag #pulmcc to educate and advocate for topics related to pulmonary, critical care, and sleep medicine. METHODS: To assess the reach of these chats, we analyzed the metrics using Symplur analytics, and compared data from each chat, as well as participant data. RESULTS: Since December 19, 2013, there have been 12 Twitter chats: six have been on critical care-related topics, four have been on pulmonary-/sleep-related topics, and two have been conducted during the CHEST annual meeting on more general topics. During these 1-h Twitter chats, there were a total of 4,212 tweets by 418 participants, resulting in 9,361,519 impressions (ie, views). There were similar numbers of participants and tweets in the three categories of Twitter chats, but there was a significantly greater reach during the more general Twitter chats conducted at the CHEST annual meeting, with 1,596,013 ± 126,472 impressions per chat session at these chats, compared with 739,203 ± 73,109 impressions per chat session during the critical care Twitter chats and 621,965 ± 123,933 impressions per chat session in the pulmonary/sleep chats. Seventy-five participants participated in two or more #pulmcc Twitter chats, and the average percent of return participants in each chat was 30% ± 7%. Most of the return participants were health-care providers. CONCLUSIONS: Twitter chats can be a powerful tool for the widespread engagement of a medical audience.

The intelligent use of digital tools and social media in practice management.

The Internet has fundamentally transformed the way patients and health-care providers communicate and interact. The use of digital tools and social media platforms, such as blogs, Facebook, Instagram, and Twitter, have empowered patients to expand their health-care knowledge and have provided practitioners with new ways to gain knowledge, lead discussions, promote causes, and build relationships with patients and other providers. In this article, we discuss the difference between digital communication, static one-way digital presence, and two-way social media connections. We also describe ways to establish and foster your digital profile, review the benefits and risks of engaging professionally in social media, and describe ways in which digital and social media tools may prove useful in both reimbursement and practice management.

H/D exchange centroid monitoring is insufficient to show differences in the behavior of protein states.

Differential hydrogen/deuterium exchange (H/DX) coupled with mass spectrometry (H/DX-MS) offers a rapid and sensitive characterization of changes in proteins following perturbations induced by changes in folding, ligand binding, oligomerization, and modification. The characterization of H/DX rates by software tools and automated data processing often relies on the centroid mass calculation and, thereby, the deuterium distribution in the mass spectra is neglected. Here we present an example demonstrating the clear limitation of using only a centroid approach to characterize the H/DX rate, in which the change in protein is not reflected as the difference in deuterium uptake based on centroid calculation.