Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-galactosidase supersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter.Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C(PKC1), gave enhanced secretion ofmost recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris.

Background The COVID-19 crisis presents new challenges and opportunities in managing alcohol use disorders, particularly for people unable to shelter in place due to homelessness or other reasons. Requiring abstinence for shelter engagement is impractical for many with severe alcohol use disorders and poses a modifiable barrier to self-isolation orders. Managed alcohol programs (MAPs) have successfully increased housing adherence for those with physical alcohol dependence in Canada, but to our knowledge, they have not been implemented in the United States. To avoid life-threatening alcohol withdrawal syndromes and to support adherence to COVID-19 self-isolation and quarantine orders, MAPs were piloted by the public health departments of San Francisco and Alameda counties. Development of MAPs We describe implementation of a first-in-the-nation alcohol use disorder intervention of a MAP that emerged at three public health isolation settings within San Francisco and Alameda counties in California. All three interventions utilized a similar process to develop the protocol and implement the MAP that included identification of champions for system-level advocacy and engagement of stakeholders. Implementation of MAPs We describe the creation and implementation of the distinct protocols. We provide examples of iterative changes to workflow processes and key lessons learned pertaining to protocol development, acceptability by stakeholders, alcohol procurement, documentation, and assessment. We discuss safety considerations, noting that there were no deaths or serious adverse events in any of the patients of the MAP during the 2-month implementation period. Conclusions MAP pilots have been implemented in the US to aid adherence to isolation and quarantine setting guidelines. Lessons learned provide a foundation for their expansion as a recognized public health intervention for individuals with severe alcohol use disorders who are unable to stabilize within existing care systems. Based on the success of MAP implementation, efforts are under way to investigate alcohol management in homeless populations more broadly.

Objective. To describe a faculty-student collaborative model and its outcomes on teaching, service, and scholarship. Design. A Medicare Part D elective course was offered that consisted of classroom and experiential learning where pharmacy students participated in community outreach events to assist Medicare beneficiaries with Part D plan selection. The course training was expanded to include medication therapy management (MTM) and the administration of immunizations. At the completion of the course, students collaborated with faculty members on research endeavors. Evaluation. During the first 6 years of this course, the class size more than doubled from 20 to 42 students, and all students participating in the course met the IPPE requirements for community outreach. Over that same period, the number of beneficiaries receiving assistance with their Part D plan grew from 72 to 610; and with the help of students starting in 2011, faculty members had 28 poster presentations at national conferences, 7 invited podium presentations at national/international meetings, and published 8 manuscripts in peer-reviewed journals. Conclusion. Through collaborative efforts, this model took an elective course and provided classroom and experiential learning for students, needed health services for the community, and opportunities to pursue wide ranging research projects for faculty members and students.

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5′ untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5′UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5′UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5′UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5′UTR optimized for a higher level of expression may be challenging.

The human secretory leukocyte protease inhibitor (SLPI) is an 11.7 kD cysteine-rich protein that has been shown to possess anti-protease, anti-inflammatory, and antimicrobial properties. By using a Pichia pastoris strain that overproduces protein disulfide isomerase (PDI), we obtained greater than fivefold higher levels of SLPI than in strains expressing normal levels of PDI and containing multiple copies of the SLPI gene. Elevated levels of PDI also enhanced the specific activity of the secreted SLPI by helping it achieve a proper tertiary structure. Mass spectrometry analysis indicated a greater number of disulfide bonds in the SLPI produced by the PDI overexpresssion strain compared to the SLPI produced in strains with normal PDI levels. Although others have utilized a similar strategy to increase yield, we believe that this is the first example of PDI overexpression being demonstrated to enhance the folding and thus increase the biological activity of a protein produced in the yeast Pichia pastoris.