Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage, A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. the mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. the variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. the new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (K-i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase, the variant LDTI-10T binds to thrombin but does not inhibit it, the relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V), the data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. (C) 1999 Federation of European Biochemical Societies.UNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUniv Munich, Klinikum Innenstadt, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, D-8000 Munich, GermanyUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilWeb of Scienc

Enhanced attraction between drops carrying fluctuating charge distributions

The electrostatic force between conductive spheres is always attractive at small separations, irrespective of their mean charge, when the charge on the spheres is constant. In many situations, the charge may not be fixed, such as for water drops in the natural atmosphere which vary in size and charge. We show that the attractive force between charged conductive spheres increases with increasing charge variance. The importance of this unrecognized electrostatic effect between water drops is evaluated for its potential to enhance rain formation

The Sexual Health Needs of Adolescent Boys Involved in a Pregnancy

Objectives: Little is known about fatherhood in middle adolescence. In order to better understand their sexual health needs, we describe relationship characteristics, perception of masculinity and associated STI risk behaviors in a community-based sample of urban middle adolescent boys who have fathered a child or been involved with a pregnancy. Methods: We employed venue-based sampling to recruit 339 boys (14-17 years old) in neighborhoods with high STI prevalence. We administered a brief survey on sexual, relationship and pregnancy history, STI risk, juvenile justice involvement, and masculinity. Results: Fifteen percent had either fathered a child or been involved with a pregnancy. In multivariate analysis, controlling for age and ethnicity, adolescent fathers were more likely to be involved with juvenile justice and engage in STI risk behaviors. These included condom non-use and partner checking a cell phone. Although of borderline significance, older partners, past STI testing, and drug or alcohol use at last sex improved model fit. Conclusion: Adolescent fathers have distinct relational and sexual health needs. Their specific needs should be targeted by prevention programs. Implications and Contribution: Most research on young men involved in pregnancy is with older adolescents/young adults, and in clinical or institutional settings. Using community engagement and venue-based sampling, this study describes sexual behaviors, masculinity, and relationship characteristics among 14-17 year old boys who have caused a pregnancy. Findings identify their distinct sexual health needs

Replacement therapy for bleeding episodes in factor VII deficiency: A prospective evaluation

Patients with inherited factor VII (FVII) deficiency display different clinical phenotypes requiring ad hoc management. This study evaluated treatments for spontaneous and traumatic bleeding using data from the Seven Treatment Evaluation Registry (STER). One-hundred one bleeds were analysed in 75 patients (41 females; FVII coagulant activity <1-20%). Bleeds were grouped as haemarthroses (n=30), muscle/subcutaneous haematomas (n=16), epistaxis (n=12), gum bleeding (n=13), menorrhagia (n=16), central nervous system (CNS; n=9), gastrointestinal (GI; n=2) and other (n=3). Of 93 evaluable episodes, 76 were treated with recombinant, activated FVII (rFVIIa), eight with fresh frozen plasma (FFP), seven with plasma-derived FVII (pdFVII) and two with prothrombin-complex concentrates. One-day replacement therapy resulted in very favourable outcomes in haemarthroses, and was successful in muscle/subcutaneous haematomas, epistaxis and gum bleeding. For menorrhagia, single- or multiple-dose schedules led to favourable outcomes. No thrombosis occurred; two inhibitors were detected in two repeatedly treated patients (one post-rFVIIa, one post-pdFVII). In FVII deficiency, most bleeds were successfully treated with single 'intermediate' doses (median 60 µg/kg) of rFVIIa. For the most severe bleeds (CNS, GI) short- or long-term prophylaxis may be optimalPatients with inherited factor VII (FVII) deficiency display different clinical phenotypes requiring ad hoc management. This study evaluated treatments for spontaneous and traumatic bleeding using data from the Seven Treatment Evaluation Registry (STER). One-hundred one bleeds were analysed in 75 patients (41 females; FVII coagulant activity <1-20%). Bleeds were grouped as haemarthroses (n=30), muscle/subcutaneous haematomas (n=16), epistaxis (n=12), gum bleeding (n=13), menorrhagia (n=16), central nervous system (CNS; n=9), gastrointestinal (GI; n=2) and other (n=3). Of 93 evaluable episodes, 76 were treated with recombinant, activated FVII (rFVIIa), eight with fresh frozen plasma (FFP), seven with plasma-derived FVII (pdFVII) and two with prothrombin-complex concentrates. One-day replacement therapy resulted in very favourable outcomes in haemarthroses, and was successful in muscle/subcutaneous haematomas, epistaxis and gum bleeding. For menorrhagia, single- or multiple-dose schedules led to favourable outcomes. No thrombosis occurred; two inhibitors were detected in two repeatedly treated patients (one postrFVIIa, one post-pdFVII). In FVII deficiency, most bleeds were successfully treated with single 'intermediate' doses (median 60 μg/kg) of rFVIIa. For the most severe bleeds (CNS, GI) short- or long-term prophylaxis may be optimal. © Schattauer 2013

Gang Exposure and Pregnancy Incidence among Female Adolescents in San Francisco: Evidence for the Need to Integrate Reproductive Health with Violence Prevention Efforts

Among a cohort of 237 sexually active females aged 14–19 recruited from community venues in a predominantly Latino neighborhood in San Francisco we examined the relationship between gang exposure and pregnancy incidence over two years of follow-up. Using discrete-time survival analysis we investigated whether individual and partner gang membership were associated with pregnancy incidence and determined whether partnership characteristics, contraceptive behaviors and pregnancy intentions mediated the relationship between gang membership and pregnancy. Pregnancy incidence was determined by urine-based testing and self-report. Seventy-seven percent of participants were Latinas, with one in five born outside the U.S. One-quarter (27.4%) became pregnant over follow-up. Participants’ gang membership had no significant effect on pregnancy incidence (Hazard Ratio (HR)=1.25; 95% confidence interval (CI): 0.54, 3.45); however, having partners who were in gangs was associated with pregnancy (HR=1.90; 95% CI: 1.09, 3.32). Perceived male partner’s pregnancy intentions and having a partner in detention each mediated the effect of partner’s gang membership on pregnancy risk. Increased pregnancy incidence among young women with gang-involved partners highlights the importance of integrating reproductive health prevention into programs for gang-involved youth. In addition, high pregnancy rates indicate a heightened risk for sexually transmitted infections

Fabrication of metallic patterns by microstencil lithography on polymer surfaces suitable as microelectrodes in integrated microfluidic systems

Microstencil lithography, i.e. local deposition of micrometer scale patterns through small shadow masks, is a promising method for metal micropattern definition on polymer substrates that cannot be structured using organic-solvent-based photoresist technology. We propose to apply microstencil lithography to fabricate microelectrodes on flat and 3D polymer substrates, such as PMMA or SU-8, which form parts of microfluidic systems with integrated microelectrodes. Microstencil lithography is accompanied by two main issues when considered for application as a low-cost, reproducible alternative to standard photolithography on polymer substrates. In this paper we assess in detail (i) the reduction of aperture size (clogging) after several metal evaporation steps and corresponding change of deposited pattern size and (ii) loss in the resolution (blurring) of the deposited microstructures when there is a several micrometers large gap between the stencil membrane and the substrate. The clogging of stencil apertures induced by titanium and copper evaporation was checked after each evaporation step, and it was determined that approximately 50% of the thickness of the evaporated metals was deposited on the side walls of the stencil apertures. The influence of a gap on the deposited structures was analyzed by using 18 um thick SU-8 spacers placed between the microstencil and the substrate. The presence of an 18 um gapmade the deposited structures notably blurred. The blurring mechanism of deposited structures is discussed based on a simplified geometrical model. The results obtained in this paper allow assessing the feasibility of using stencil-based lithography for unconventional surface patterning, which shows the limits of the proposed method, but also provides a guideline on a possible implementation for combined polymer-electrode microsystems, where standard photoresist technology fails

Variation in carbon isotope discrimination in Cleistogenes squarrosa (Trin.) Keng: patterns and drivers at tiller, local, catchment, and regional scales

Understanding the patterns and drivers of carbon isotope discrimination in C4 species is critical for predicting the effects of global change on C3/C4 ratio of plant community and consequently on ecosystem functioning and services. Cleistogenes squarrosa (Trin.) Keng is a dominant C4 perennial bunchgrass of arid and semi-arid ecosystems across the Mongolian plateau of the Eurasian steppe. Its carbon isotope discrimination (13Δ) during photosynthesis is relatively large among C4 species and it is variable. Here the 13Δ of C. squarrosa and its potential drivers at a nested set of scales were examined. Within cohorts of tillers, 13Δ of leaves increased from 5.1‰ to 8.1‰ from old to young leaves. At the local scale, 13Δ of mature leaves varied from 5.8‰ to 8.4‰, increasing with decreasing grazing intensity. At the catchment scale, 13Δ of mature leaves varied from 6.2‰ to 8.5‰ and increased with topsoil silt content. At the regional scale, 13Δ of mature leaves varied from 5.5‰ to 8.9‰, increasing with growing-season precipitation. At all scales, 13Δ decreased with increasing leaf nitrogen content (Nleaf). Nleaf was positively correlated with grazing intensity and leaf position along tillers, but negatively correlated with precipitation. The presence of the correlations across a range of different environmental contexts strongly implicates Nleaf as a major driver of 13Δ in C. squarrosa and, possibly, other C4 species