Evaluating Mobile Apps Targeting Older Adults: Descriptive Study.

BACKGROUND: Smartphone use has increased dramatically and, in parallel, a market for mobile apps, including health apps, has emerged. The business model of targeted mobile app advertisements allows for the collection of personal and potentially sensitive information, often without user knowledge. Older adults comprise a rapidly growing demographic that is potentially vulnerable to exploitation by those accessing data collected via these apps. OBJECTIVE: This research examined apps that claimed to be useful to older adults with a goal of (1) classifying the functionality of each app, (2) identifying whether a privacy policy existed and was accessible, and (3) evaluating evidence that could support claims of value to older adults. METHODS: An environmental scan was conducted using the Google search engine and typing "apps for older adults." The first 25 sites that this search returned comprised the primary data for this study. Data were organized by descriptive features of purpose (eg, health, finance, and utility), the existence of an electronically accessible privacy policy, price, and evidence supporting each recommended mobile app. RESULTS: A total of 133 mobile apps were identified and promoted as being the best "apps for older adults." Of these 133 mobile apps, 83% (n=110) included a privacy policy. Fewer apps designated in the "medical" category included a privacy policy than those classified otherwise. CONCLUSIONS: The results suggest that most mobile apps targeting older adults include a privacy policy. Research is needed to determine whether these privacy policies are readable, succinct, and incorporate accessible data use and sharing practices to mitigate potential risks, particularly when collecting potentially sensitive health information.

Directed evolution provides insight into conformational substrate sampling by SrtA.

The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme's inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.

Correction: Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin.

[This corrects the article DOI: 10.1371/journal.pone.0163732.].

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

The Evolutionary Loss of RNAi Key Determinants in Kinetoplastids as a Multiple Sporadic Phenomenon.

We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.

Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin.

Functional cross-talk between Tie2 and Integrin signaling pathways is essential to coordinate endothelial cell adhesion and migration in response to the extracellular matrix, yet the mechanisms behind this phenomenon are unclear. Here, we examine the possibility that receptor cross-talk is driven through uncharacterized Tie-integrin interactions on the endothelial surface. Using a live cell FRET-based proximity assay, we monitor Tie-integrin receptor recognition and demonstrate that both Tie1 and Tie2 readily associate with integrins α5ß1 and αVß3 through their respective ectodomains. Although not required, Tie2-integrin association is significantly enhanced in the presence of the extracellular component and integrin ligand fibronectin. In vitro binding assays with purified components reveal that Tie-integrin recognition is direct, and further demonstrate that the receptor binding domain of the Tie2 ligand Ang-1, but not the receptor binding domain of Ang-2, can independently associate with α5ß1 or αVß3. Finally, we reveal that cooperative Tie/integrin interactions selectively stimulate ERK/MAPK signaling in the presence of both Ang-1 and fibronectin, suggesting a molecular mechanism to sensitize Tie2 to extracellular matrix. We provide a mechanistic model highlighting the role of receptor localization and association in regulating distinct signaling cascades and in turn, the angiogenic switch.

Over-expression of secreted proteins from mammalian cell lines.

Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats.

Tie2 and Eph receptor tyrosine kinase activation and signaling.

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.

Structural basis for angiopoietin-1-mediated signaling initiation.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

Imaging protein-protein interactions in vivo.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET). Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.

The Tie family of endothelial-specific receptor tyrosine kinases is essential for cell proliferation, migration, and survival during angiogenesis. Despite considerable similarity, experiments with Tie1- or Tie2-deficient mice highlight distinct functions for these receptors in vivo. The Tie2 receptor is further unique with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists; angiopoietin-1 and -4 are constitutive agonists. To address the role of Tie1 in angiopoietin-mediated Tie2 signaling and determine the basis for the behavior of the individual angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and -2 localization and association. We provide evidence for Tie1-Tie2 complex formation on the cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by angiopoietin-1 and -2. Based on the available data, we propose a unified model for angiopoietin-induced Tie2 signaling.

Tongue piercing by a Yogi: QEEG observations.

: This study reports on the QEEG observations recorded from a yogi during tongue piercing in which he demonstrated voluntary pain control. The QEEG was recorded with a Lexicor 1620 from 19 sites with appropriate controls for impedence and artifacts. A neurologist read the data for abnormalities and the QEEG was analyzed by mapping, single and multiple hertz bins, coherence, and statistical comparisons with a normative database. The session included a meditation baseline and tongue piercing. During the meditative baseline period the yogi's QEEG maps suggesting that he was able to lower his brain activity to a resting state. This state showed a predominance of slow wave potentials (delta) during piercing and suggested that the yogi induced a state that may be similar to those found when individuals are under analgesia. Further research should be conducted with a group of individuals who demonstrate exceptional self-regulation to determine the underlying mechanisms, and whether the skills can be used to teach others how to manage pain.

Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells.

The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high-efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X-ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency.

Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex.

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.

Palladium-catalyzed cross-coupling of B-benzyl-9-borabicyclo[3.3.1]nonane to furnish methylene-linked biaryls.

[reaction: see text] Benzylboranes are noticeably uncommon partners within Suzuki-Miyaura coupling reactions. B-Benzyl-9-BBN was successfully coupled to a range of aryl/heteroaryl bromides, chlorides, and triflates to give pharmacologically important methylene-linked biaryl structures. Activated, deactivated, and sterically hindered substrates were successfully coupled in high yield using Pd(PPh(3))(4) or Pd(OAc)(2) with SPhos as the catalyst system.

Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans.

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling.

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.

Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition.

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.