Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Genetic basis for probiotic yeast phenotypes revealed by nanopore sequencing.

Probiotic yeasts are emerging as preventative and therapeutic solutions for disease. Often ingested via cultured foods and beverages, they can survive the harsh conditions of the gastrointestinal tract and adhere to it, where they provide nutrients and inhibit pathogens like Candida albicans. Yet, little is known of the genomic determinants of these beneficial traits. To this end, we have sequenced 2 food-derived probiotic yeast isolates that mitigate fungal infections. We find that the first strain, KTP, is a strain of Saccharomyces cerevisiae within a small clade that lacks any apparent ancestry from common European/wine S. cerevisiae strains. Significantly, we show that S. cerevisiae KTP genes involved in general stress, pH tolerance, and adherence are markedly different from S. cerevisiae S288C but are similar to the commercial probiotic yeast species S. boulardii. This suggests that even though S. cerevisiae KTP and S. boulardii are from different clades, they may achieve probiotic effect through similar genetic mechanisms. We find that the second strain, ApC, is a strain of Issatchenkia occidentalis, one of the few of this family of yeasts to be sequenced. Because of the dissimilarity of its genome structure and gene organization, we infer that I. occidentalis ApC likely achieves a probiotic effect through a different mechanism than the Saccharomyces strains. Therefore, this work establishes a strong genetic link among probiotic Saccharomycetes, advances the genomics of Issatchenkia yeasts, and indicates that probiotic activity is not monophyletic and complimentary mixtures of probiotics could enhance health benefits beyond a single species.

Publicly Available hiPSC Lines with Extreme Polygenic Risk Scores for Modeling Schizophrenia.

Schizophrenia (SZ) is a common and debilitating psychiatric disorder with limited effective treatment options. Although highly heritable, risk for this polygenic disorder depends on the complex interplay of hundreds of common and rare variants. Translating the growing list of genetic loci significantly associated with disease into medically actionable information remains an important challenge. Thus, establishing platforms with which to validate the impact of risk variants in cell-type-specific and donor-dependent contexts is critical. Towards this, we selected and characterized a collection of 12 human induced pluripotent stem cell (hiPSC) lines derived from control donors with extremely low and high SZ polygenic risk scores (PRS). These hiPSC lines are publicly available at the California Institute for Regenerative Medicine (CIRM). The suitability of these extreme PRS hiPSCs for CRISPR-based isogenic comparisons of neurons and glia was evaluated across 3 independent laboratories, identifying 9 out of 12 meeting our criteria. We report a standardized resource of publicly available hiPSCs on which we hope to perform genome engineering and generate diverse kinds of functional data, with comparisons across studies facilitated by the use of a common set of genetic backgrounds.

Working with and for: student advocates' experience of relationship-centered advocacy with low-income women.

This study describes a qualitative study of student advocates' experiences of their work with low-income women struggling with symptoms of depression. Using an advocacy model called Relationship-Centered Advocacy, these 1st-year counseling psychology graduate students worked intensively with their "partners" for 9 months. Advocate-partner teams met together each week, developing collaborative relationships and addressing the women's emotional and material needs in integrated ways. Using qualitative content analysis of participant interviews and journal entries, this study describes emergent themes involving negotiating the advocacy relationship, insider-outsider dynamics, responding to perceptions of privilege and disparity, and gaining professional and personal insights. It concludes with a discussion of the practice and research implications, highlighting the possibilities of Relationship-Centered Advocacy as a new training tool.

A theoretical framework for understanding help-seeking processes among survivors of intimate partner violence.

This paper suggests a conceptual framework for understanding the processes of help-seeking among survivors of intimate partner violence (IPV). A cognitive theory from general literature on help-seeking in "stigmatizing" situations suggests three relevant processes or stages of seeking help in the IPV context: defining the problem, deciding to seek help, and selecting a source of support. Individual, interpersonal, and sociocultural factors that influence decision-making at each of these stages are discussed and illustrated with case examples.