Bottling the “Secret Sauce”: Capturing the Heart of the East End at Peter Paul Development Center

Peter Paul Development Center (Peter Paul) submitted a Request for Assistance (RFA) to obtain support from a Virginia Commonwealth University Doctorate of Educational Leadership (EdD) Capstone team with documenting programming and principles and evaluating them consistently. To address this request, the team analyzed the context and problem of practice through document review, empathy interviews, partner discussions and site visits. The literature review helped the team design and conduct research, fieldwork, and data analysis to support Peter Paul in standardizing procedures and programs, fostering a culture of institutional knowledge, and effectively communicating impact to stakeholders. The team used a Convergent Parallel mixed method research design. The team conducted interviews, site observations, a culture assessment, and a document review. Findings center around a need for increased communication about programs, institutional knowledge, and data demonstrating their impact. By examining the role of these elements in reshaping organizational identity and creating a sustainable work model, the study offers practical insights for Peter Paul’s leaders on how to define their identity or “secret sauce,” operational efficiency, and long-term sustainability in a competitive nonprofit landscape. Keywords: culture, impact, institutional knowledge, knowledge management, nonprofit, organizational culture, organizational effectiveness, organizational identity, standardizing programs and procedures, “secret sauce,” sustainabilit

Utilization of the Mating Scaffold Protein in the Evolution of a New Signal Transduction Pathway for Biofilm Development

Among the hemiascomycetes, only Candida albicans must switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does in Saccharomyces cerevisiae, Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization

Evolutionary Reshaping of Fungal Mating Pathway Scaffold Proteins

Scaffold proteins play central roles in the function of many signaling pathways. Among the best-studied examples are the Ste5 and Far1 proteins of the yeast Saccharomyces cerevisiae. These proteins contain three conserved modules, the RING and PH domains, characteristic of some ubiquitin-ligating enzymes, and a vWA domain implicated in protein-protein interactions. In yeast, Ste5p regulates the mating pathway kinases while Far1p coordinates the cellular polarity machinery. Within the fungal lineage, the Basidiomycetes and the Pezizomycetes contain a single Far1-like protein, while several Saccharomycotina species, belonging to the CTG (Candida) clade, contain both a classic Far1-like protein and a Ste5-like protein that lacks the vWA domain. We analyzed the function of C. albicans Ste5p (Cst5p), a member of this class of structurally distinct Ste5 proteins. CST5 is essential for mating and still coordinates the mitogen-activated protein (MAP) kinase (MAPK) cascade elements in the absence of the vWA domain; Cst5p interacts with the MEK kinase (MEKK) C. albicans Ste11p (CaSte11p) and the MAPK Cek1 as well as with the MEK Hst7 in a vWA domain-independent manner. Cst5p can homodimerize, similar to Ste5p, but can also heterodimerize with Far1p, potentially forming heteromeric signaling scaffolds. We found direct binding between the MEKK CaSte11p and the MEK Hst7p that depends on a mobile acidic loop absent from S. cerevisiae Ste11p but related to the Ste7-binding region within the vWA domain of Ste5p. Thus, the fungal lineage has restructured specific scaffolding modules to coordinate the proteins required to direct the gene expression, polarity, and cell cycle regulation essential for mating

Many roads to symmetry breaking: Molecular mechanisms and theoretical models of yeast cell polarity

Mathematical modeling has been instrumental in identifying common principles of cell polarity across diverse systems. These principles include positive feedback loops that are required to destabilize a spatially uniform state of the cell. The conserved small G-protein Cdc42 is a master regulator of eukaryotic cellular polarization. Here we discuss recent developments in studies of Cdc42 polarization in budding and fission yeasts and demonstrate that models describing symmetry-breaking polarization can be classified into six minimal classes based on the structure of positive feedback loops that activate and localize Cdc42. Owing to their generic system-independent nature, these model classes are also likely to be relevant for the G-protein–based symmetry-breaking systems of higher eukaryotes. We review experimental evidence pro et contra different theoretically plausible models and conclude that several parallel and non–mutually exclusive mechanisms are likely involved in cellular polarization of yeasts. This potential redundancy needs to be taken into consideration when interpreting the results of recent cell-rewiring studies

Applying an Integrative Heritable Component Approach to the Identification of Highly Heritable Traits in Diseases with Complex Etiology

Despite numerous recent genome-wide association studies on alcohol use, progress in identifying genetic associations for Alcohol Use Disorder (AUD) has been limited due to the extensive heterogeneity associated with this disorder in terms of clinical manifestations, underlying genetics, and environmental factors. To address this challenge, we propose the use of a novel statistical approach which integrates phenotypic, genotypic, and environmental data to derive disease related traits with maximized heritability. The method will be applied in two phases. In phase 1, a statistical method will be developed that aims to identify a function that leads to the identification of a trait with a maximized heritability estimate. This method will make use of a linear-mixed-model which will account for the moderating effects of environmental factors. We will further investigate the application of a constraint to the derived traits which will allow them to be interpreted as severity indexes for alcohol use disorder. In phase 2, genome-wide association studies will be conducted to identify genetic risk factors associated with the derived traits

Inositol pyrophosphates modulate S phase progression after pheromone-induced arrest in Saccharomyces cerevisiae

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest

Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gβγ freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic α-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388–518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gβγ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK)