The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.
Hantavirus Cardiopulmonary Syndrome (HCPS) due to Andes virus (ANDV) is endemic in Chile and Argentina and currently demonstrates a case-fatality rate of 37% in humans. By contrast to the chronically infected rodents, it is believed that ANDV in humans is cleared during the acute phase. Moreover, to date, both magnitude and quality of human T-cell responses during ANDV infection and clearance are unknown. Using IFN-gamma and granzyme B ELISPOT assays as well as flow cytometry, we prospectively studied the ANDV-specific T-cell responses in a 56-year-old convalescing survivor of severe HCPS, whose blood cells remained PCR-positive for ANDV-RNA until day 53 after hospital admission, that is, 67 days after infection and 42 days after discharge. PCR-negativity was closely related to the increase and function of (Gn(46-60))-specific IFN-gamma(+) granzyme B(+) CD8(+) T-cells, but not to neutralizing antibody titers. Concurrently, the phenotype of CD45RA(+)CCR7(-) Gn(46-60)-specific T-cells shifted from a CD28(-)CD27(+) "intermediate" to a CD28(-) CD27(-) "late" effector memory beyond day 53 after hospital admission. This is the first report that shows that ANDV can persist in the human hosts for more than 2 months. Moreover, the kinetics of T-cell responses during ANDV clearance may indicate a major role of T-cells for clearance of ANDV and human immunity to this pathogen.
Objective. Clinical documentation is an important element of patient care that pharmacy students traditionally learn through Subjective-Objective-Assessment-Plan (SOAP) notes. In clinical practice, pharmacists often document more succinctly both in regard to length and time; utilizing formats such as consult notes. The objective of this study was to assess consult note assignments for third-year pharmacy students (P3). Methods. Consult note assignments were implemented in a P3 skills laboratory course by converting SOAP notes to consult notes. The series began with an introduction and a practice consult note. Four graded notes were then completed throughout the semester with time allotted for writing decreasing throughout the semester. To assess the series, grades and estimated time for completion were collected for each graded note. A pre-post survey assessed student self-confidence in: 1) overall documentation, 2) specific elements of consult notes, and 3) concerns related to writing. Friedman's tests were utilized to compare grades and time. Wilcoxon Signed Rank tests were used to compare self-assessments. Results. Median consult note grades were: 92%, 88%, 80%, and 90% for each note. Median time for completion were: 75 minutes, 120 minutes, 60 minutes, and 60 minutes. Student self-confidence in writing consult notes significantly increased as did five of the six individual elements. Conclusion. The consult note assignments allowed students to practice documenting patient care in a succinct format with consideration for time efficiency. Further work should evaluate best pedagogies for teaching documentation skills and assess the impact on performance during advanced pharmacy practice experiences.
Dopamine-receptor blocking agent-associated akathisia (DRBA-A) is an adverse effect that can significantly limit the use of these important medications for the treatment of a variety of psychiatric diseases, yet there is no unifying theory regarding its pathophysiology. This knowledge gap limits clinicians’ ability to effectively manage DRBA-A and mitigate negative outcomes in an already vulnerable patient population. Based on a review of the current literature on the subject, it is hypothesized that dopaminergic and noradrenergic signaling is perturbed in DRBA-A. Accordingly, it is proposed that the optimal agent to manage this extrapyramidal symptom should increase dopamine signaling in the affected areas of the brain and counteract compensatory noradrenergic signaling via antagonism of adrenergic or serotonergic receptors.
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