Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery

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Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.Cystic Fibrosis Foundation (Research Development Program)National Center for Research Resources (U.S.) (P20RR018787-06

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs

Integrating community health assistant- driven sexual and reproductive health services in the community health system in Nyimba district in Zambia: Mapping key actors, points of integration, and conditions shaping the process

Introduction: Although large scale public sector community health worker programs have been key in providing sexual and reproductive health (SRH) services in low- and middle-income countries, their integration process into community health systems is not well understood. This study aimed to identify the conditions and strategies through which Community Health Assistants (CHAs) gained entry and acceptability into community health systems to provide SRH services to youth in Zambia. The country’s CHA program was launched in 2010. Methodology: A phenomenological design was conducted in Nyimba district. All nine CHAs deployed in Nyimba district were interviewed in-depth on their experiences of navigating the introduction of SRH services for youth in community settings, and the data obtained analyzed thematically. Results: In delivering SRH services targeting youth, CHAs worked with a range of community actors, including other health workers, safe motherhood action groups, community health workers, neighborhood health committees, teachers, as well as political, traditional and religious leaders. CHAs delivered SRH education and services in health facilities, schools, police stations, home settings, and community spaces. They used their health facility service delivery role to gain trust and entry into the community, and they also worked to build relationships with other community level actors by holding regular joint meetings, and acting as brokers between the volunteer health workers and the Ministry of Health. CHAs used their existing social networks to deliver SRH services to adolescents. By embedding the provision of information about SRH into general life skills at community level, the topic’s sensitivity was reduced and its acceptability was enhanced. Further, support from community leaders towards CHA-driven services promoted the legitimacy of providing SRH for youth. Factors limiting the acceptability of CHA services included the taboo of discussing sexuality issues, a gender discriminatory environment, competition with other providers, and challenges in conducting household visits

Recombinant antibody selections by combining phage and yeast display

n vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting. The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest. In the present chapter, we illustrate protocols to generate and enrich - using fluorescence-activated cell sorting (FACS) - yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties