Successful implementation of managed alcohol programs in the San Francisco Bay Area during the COVID-19 crisis.

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.

Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

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-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a ß-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 of most 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.

An improved method for enhanced production and biological activity of human secretory leukocyte protease inhibitor (SLPI) in Pichia pastoris.

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 overexpression 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 P. pastoris.

Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris.

The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins.

Antipsychotic Selection for Acute Agitation and Time to Repeat Use in a Psychiatric Emergency Department.

BACKGROUND: Early recognition and treatment of agitated patients is essential to avoid violence in the psychiatric emergency department (ED). Antipsychotics have established efficacy in managing agitation, yet little is known about how the choice of initial antipsychotic impacts time to repeat use and length of stay (LOS) in the psychiatric ED. OBJECTIVE: To describe the impact of initial antipsychotic selection on time to repeat use and LOS in the psychiatric ED. METHODS: A chart review identified 388 cases in which patients were administered an antipsychotic for agitation in the psychiatric ED between July 1 and August 31, 2014. Time to repeat use and LOS were compared for intramuscular (IM) haloperidol, other IM antipsychotics, and oral second-generation antipsychotics (SGAs) using the Kruskal-Wallis or Wilcoxon-Mann-Whitney test. RESULTS: Of the 388 cases, 31% (n=122) required repeat medications. Mean time to repeat use for IM haloperidol was 20.1±18.4 hours, which was not significantly different from mean time to repeat use in the groups receiving other IM antipsychotics or oral SGAs (P=0.35). The mean LOS was 29.7±28.7 hours for IM haloperidol, 30.3±36.9 hours for other IM antipsychotics, and 22.6±28.0 hours for oral SGAs. Significant differences in LOS between repeat and nonrepeat users of IM haloperidol and other IM antipsychotics were observed, but not among those who received oral SGAs. CONCLUSIONS: Mean time to repeat use ranged from 14 to 20 hours with IM haloperidol, other IM antipsychotics, and oral SGAs without significant differences in time to repeat use in the 3 different groups. Repeat users of IM antipsychotics had a significantly longer LOS in the ED compared with nonrepeat users of IM antipsychotics. However, patients who were initially administered oral SGAs did not have longer LOS in the ED even if a repeat dose was given.

A collaborative approach to combining service, teaching, and research.

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.

Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris.

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 ß-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.