Artificial mobile DNA element constructed from the EcoRI endonuclease gene.

There exist several examples of mobile group I introns. These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes. Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless recipient DNA molecule, we reasoned that it should in principle be possible to construct artificially mobile DNA sequences. We have constructed an artificial mobile element from the gene for the restriction enzyme EcoRI that is capable of site-specific insertion at rates near those of authentic mobile introns. The generality of the mobility mechanism may enable high-efficiency targeted gene replacements or disruptions in a variety of organisms.     

Mentoring Youth at High Risk: The Perspectives of Professional Mentors

Background

Youth mentoring programs rely largely on volunteers, but youth facing significant risks may be poor candidates for volunteer-based interventions. Full-time “professional” mentors in highly structured programs may be better suited to partner effectively with such youth and their families, but few studies examine professional mentoring interventions. Because of mentoring’s inherent flexibility, mentors’ role conceptualizations can profoundly influence the nature of their work. Serving as a professional mentor may have important implications for how mentors conceptualize and perform their role.

Objective

This qualitative study examined the role conceptions of professional mentors serving at-risk youth.

Methods

Semi-structured interviews with mentors were transcribed, coded, and subjected to thematic analysis.

Results

Mentors described the importance of “professionalism” in prioritizing mentoring, expending considerable effort, and performing difficult or unpleasant tasks. They reported that serving multiple children full-time enabled them to rapidly build expertise, that credibility and authority granted them because of their professional status facilitated their work across multiple key contexts, and that their expertise and long-term commitment facilitated the development of deep relationships. Mentors perceived their role as highly challenging but reported high self-efficacy. They described high multifaceted organizational support, a community for youth, and an individualized child focus.

Conclusions

A mentoring model delivered by experienced professional mentors may hold promise for working with youth at high risk. The role conceptualizations of mentors and the organizational culture within which mentors work may be important in helping youth succeed.     

Genetic variants of ApoE and ApoER2 differentially modulate endothelial function

It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE–ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte–endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2^−/− mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2^−/− mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.Cardiovascular disease risk is modified by common genetic variants of apolipoprotein E (apoE) and its receptor apolipoprotein E receptor 2 (ApoER2), which is a member of the LDL receptor family. Compared with the most common allele apoE3, individuals with the apoE4 allele have an increased risk of atherosclerosis and coronary heart disease (1, 2). The LRP8 gene, which encodes ApoER2, is a major gene locus for premature atherosclerosis and acute myocardial infarction identified in four independent human populations. In particular, homozygous carriers of the ApoER2-R952Q variant have a twofold increased risk of these conditions (3–5). ApoER2-R952Q also has an additive effect with apoE4, with the combined genotype QQ/E4 showing a 3.9-fold greater susceptibility to cardiovascular disease (5). ApoER2 polymorphism-associated risk is independent of cholesterol levels (3–5), and although apoE4 may impact LDL abundance (2), there is also evidence that apoE4-associated risk goes well beyond changes in lipoprotein status (6–9). Whereas there is considerable understanding of the biology of the apoE–ApoER2 tandem in the central nervous system and in Alzheimer’s disease (10), the basis for the cardiovascular impact of the receptor and apoE variants remains unclear.Our prior work demonstrated that ApoER2 is expressed in endothelial cells, where it plays a critical role in the pathogenesis of the antiphospholipid syndrome (APS) (11). The receptor is enriched in caveolae/lipid rafts in which signaling molecules regulating endothelial NOS (eNOS) are compartmentalized (12, 13). We now know that in APS, antiphospholipid antibody recognition of the cell surface protein β2-GPI on endothelial cells promotes β2-GPI dimerization and interaction with the extracellular domain of ApoER2, causing the activation of PP2A and eNOS antagonism. The resulting decrease in bioavailable NO underlies APS-related thrombosis (11). However, the normal function of the receptor in endothelium, and whether and how it modulates apoE actions on endothelium, are unknown.In addition to regulating thrombogenesis, eNOS-derived NO plays a major role in cardiovascular protection via promotion of the integrity of the endothelial cell monolayer and attenuation of endothelial cell–leukocyte adhesion (13). Recognizing that eNOS enzymatic activity is both positively and negatively modulated by signaling molecules in endothelial caveolae/lipid rafts (14), to better understand the biology of ApoER2 in endothelium we hypothesized that apoE3 binding to the receptor activates eNOS. Experiments were performed in cell culture and in mice to test this hypothesis and to determine whether genetic variants in apoE or ApoER2 disrupt this process and thereby adversely impact endothelial function.     

Order and disorder control the functional rearrangement of influenza hemagglutinin

Influenza hemagglutinin (HA), a homotrimeric glycoprotein crucial for membrane fusion, undergoes a large-scale structural rearrangement during viral invasion. X-ray crystallography has shown that the pre- and postfusion configurations of HA₂, the membrane-fusion subunit of HA, have disparate secondary, tertiary, and quaternary structures, where some regions are displaced by more than 100 Å. To explore structural dynamics during the conformational transition, we studied simulations of a minimally frustrated model based on energy landscape theory. The model combines structural information from both the pre- and postfusion crystallographic configurations of HA₂. Rather than a downhill drive toward formation of the central coiled-coil, we discovered an order-disorder transition early in the conformational change as the mechanism for the release of the fusion peptides from their burial sites in the prefusion crystal structure. This disorder quickly leads to a metastable intermediate with a broken threefold symmetry. Finally, kinetic competition between the formation of the extended coiled-coil and C-terminal melting results in two routes from this intermediate to the postfusion structure. Our study reiterates the roles that cracking and disorder can play in functional molecular motions, in contrast to the downhill mechanical interpretations of the “spring-loaded” model proposed for the HA₂ conformational transition.Hemagglutinin (HA) is a viral receptor-binding and membrane-fusion glycoprotein involved in the invasion of influenza virions into host cells (1). Structural rearrangements of HA during membrane fusion are crucial for the delivery of the viral genome. The postfusion conformation of HA shows considerable similarity to other viral fusion proteins and eukaryotic membrane receptors involved in intracellular vesicle trafficking (2), suggesting there may be common mechanisms in the function of these proteins. Therefore, HA may serve as a model system, allowing characterization of the molecular and energetic details that underlie its conformational transition to provide insights into general principles of membrane fusion (3).HA is a homotrimer consisting of two domains connected by disulfide bonds (4): a globular receptor binding domain (HA₁), and a coiled-coil membrane-fusion domain anchored to the viral membrane (HA₂). Recognized by the sialic acid receptor of a host cell, the intact virus enters the cell via endocytosis. Low pH in a late endosome then induces the dissociation of HA₁ from HA₂ (1) and an irreversible conformational transition of HA₂. Experimentally, this conformational change can be triggered by either low pH, high temperature, or urea denaturation (5).Structures of HA in pre- and postfusion pH conformations have been solved by X-ray crystallography. The structure of the prefusion ectodomain contains both HA₁ and HA₂, and was purified from influenza virions (4). A postfusion conformation of HA₁ and HA₂ were obtained from prefusion viral HA that was sequentially treated with low pH and trypsin (6, 7). Comparison of these two structures shows no structural changes in HA₁, but a major rearrangement in HA₂, including secondary, tertiary, and quaternary structural changes. The N-terminal domain of HA₂, initially adjacent to the transmembrane region in the prefusion configuration, undergoes a large movement (over 100 Å) during the transition. The C-terminal domain of HA₂ changes from a globular structure to three extended loops packed against the central coiled-coil.Although experiments have probed the fusion mechanism through mutation (8) and provided measures of fusion kinetics (9), there is a lack of structural information about how HA₂ transitions from the prefusion to postfusion conformations. Theoretical models have been suggested to describe the fusion mechanism based on the available experimental kinetic data (10–12). However, because of the large scale of the HA₂ rearrangement, only limited computational techniques, such as targeted molecular dynamics (13), have been applied to study the molecular details of the transition.In this study we applied the principles of the energy landscape theory as developed in the context of protein folding (14–16) to examine structural details of the HA₂ conformational transition. We used a structure-based model (SBM) (17, 18) built with a dual-funneled landscape (19, 20) that has both the pre- and postfusion structures as explicit minima. The HA₂ landscape has at least two competing basins of attraction, corresponding to the pre- and postfusion structures of HA₂, respectively. HA₁ dissociation sterically enables HA₂ to explore beyond the prefusion local free-energy minimum and to diffuse toward the postfusion configuration. The long-length scale and extensive shuffling of secondary and tertiary structures is reminiscent of protein folding, but distinct in that both ends of the HA₂ transition can be described by ensembles of structurally similar configurations. Just as in protein folding, there may be free-energy barriers and structural intermediates along the HA₂ transition caused by the imperfect cancellation of energy and entropy. These intermediate ensembles may be interesting candidates for drug design to inhibit HA function.Previously, a “spring-loaded” model has been applied to describe the mechanism for the HA₂ transition (21). This model suggested a downhill mechanical transition of the N-terminal region of HA₂ into an ordered helical structure that orients the fusion peptides away from the virus and toward the host membrane. Our simulations expand this view by showing that the conformational change of the N-terminal domains is associated with an entropic barrier and the unfolding of the C-terminal region is associated with the major energetic barrier during the HA₂ conformational transition. Kinetic competition between these two events creates a long-lived metastable intermediate that allows for two dominant routes. The first route (the “sequential route”) resembles the spring-loaded model, and the second route (the “cooperative route”) involves cooperative interactions between the N-terminal and C-terminal domains in forming the central coiled-coil. The presence of these distinct routes suggests multiple mechanisms for HA₂ rearrangement and membrane fusion.     

Transforming Growth Factor β1 Regulates Tissue Inhibitor of Metalloproteinases—1 Expression in Differentiated Human Articular Chondrocytes

Objective. To investigate the role of interleukin-6 (IL-6) and transforming growth factor β1 (TGF β1) in the regulation of tissue inhibitor of metalloproteinases—1 (TIMP-1) synthesis in human articular chondrocytes. Methods. Articular cartilage was obtained from human knee joints 24 hours after death. Chondrocytes were isolated by collagenase digestion and embedded in low-gelling-temperature agarose. After stimulation by cytokines, total RNA was isolated and analyzed by Northern blotting. TIMP-1 protein levels were determined using a competitive enzyme-linked immunosorbent assay. Results. Human chondrocytes in agarose culture expressed messenger RNA (mRNA) for the IL-6 receptor (gp80) and its signal-transducing subunit gp130. In contrast to the findings in a previous study, IL-6 did not stimulate TIMP-1 expression in these cells, whereas TGF β1 was an important inducer of TIMP-1 mRNA and protein synthesis. Conclusion. Our findings suggest that TGF β1 has a protective effect on the extracellular matrix of human articular chondrocytes.     

Photocatalysis of nanocomposite titania–natural silica as antibacterial against Staphylococcus aureus and Pseudomonas aeruginosa

The entries of pathogenic bacteria into the human body remain a severe problem to health that can be prevented using antibacterial agents. Meanwhile, the photocatalytic technique using semiconductor nanocomposite TiO₂–SiO₂ has great potential as an antibacterial method. In order to utilize natural resources, SiO₂ supporting materials are obtained from the extraction of beach sand due to the high silica content. Therefore, this study aims to synthesize a nanocomposite of TiO₂ with SiO₂ extracted from beach sand as an antibacterial agent against Staphylococcus aureus and Pseudomonas aeruginosa. The antibacterial activity test used the dilution and optical density method. Based on XRD analysis, the crystals of TiO₂ in the synthesized composites showed a more dominant anatase structure. Furthermore, Ti–O–Si bonds were identified from the IR spectrum, which showed the interaction between TiO₂ and SiO₂. In addition, SEM-EDX results showed agglomerated spherical particles with a TiO₂–SiO₂ nanocomposite particle size of 40–107 nm. The best antibacterial activity was demonstrated by the 1 : 0.5 TiO₂–SiO₂ nanocomposite, with inactivation percentages of S. aureus and P. aeruginosa of 98.69% and 97.44%, respectively.

TiO₂ material is composited with silica obtained from natural sand with indirect sonochemistry method. The addition of SiO₂ increase the photocatalyst activity of TiO₂ as an antibacterial against S. aureus and P. aeruginosa.