Elevating the Importance of Local Elections.

This Viewpoint discusses the reasons why local elections are important and the ways that health care professionals can get involved.

A split ribozyme that links detection of a native RNA to orthogonal protein outputs.

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.

Enhancing Physician Advocacy Through Collaboration With Government Relations: Report From the Health Policy Scholars Program.

OBJECTIVE: To explore barriers, facilitators, and benefits of collaboration between academic pediatricians and institutional offices of government relations (OGR) to enhance policy advocacy efforts. METHODS: The Academic Pediatric Associations' Health Policy Scholars met with the government relations team in their affiliated institutions as part of their experiential learning curriculum. Afterward, they submitted written reflections, which were coded and analyzed using inductive qualitative content analysis to identify key themes. RESULTS: Reflections were completed by 21 of 23 (91.3%) participants. Most participants (76.2%) were faculty at free-standing children's hospitals and had been at their institutions <5 years (52.3%) or 5 to 10 years (33.3%). Institutional OGR structure varied widely and not all institutions had well-defined priorities. Key themes of the reflections included that OGRs often had dynamic priorities and fiscal considerations frequently took precedence. Barriers to physician involvement with OGR are often related to difficulty identifying the correct staff contact and not having the time and support for advocacy work. Facilitators included leveraging existing relationships and collaborations, including those of peer or mentor connections to the OGR staff. Anticipated benefits to both OGR and physicians included improved knowledge of advocacy opportunities, enhanced advocacy efforts leveraging physicians' expertise and patient stories, and message alignment and amplification of physician and institutional advocacy work. CONCLUSIONS: Collaboration between physician-advocates and institutional OGR is feasible and, with orientation and mentorship, may facilitate improved physician and institutional policy advocacy efforts. Supporting this type of collaboration may enhance physician and institutional advocacy on behalf of their shared patients and communities.

Demystifying the Op-Ed. A Novel Group Writing Workshop to Improve Upon Existing Pediatric Advocacy Training.

An op-ed writing workshop utilizing a group compilation exercise increases participant self-reported comfort in writing op-eds and has led to published op-eds. An experiential op-ed writing workshop could be incorporated into advocacy curricula in pediatric residency programs.

Changing Who Has a Seat and Voice at the Table: How the Academic Pediatric Association is Responding to Systemic Racism.

The Academic Pediatrics Association has taken multiple steps over the last 2 years to incorporate the lenses of anti-racism and social justice into our mission and work. In this commentary, we discuss the creation and work of the Anti-Racism and Diversity Task Force, which was charged by the Academic Pediatrics Association's Board of Directors with identifying strategies to promote anti-racism and advance the diversity, equity and inclusion agenda.

Optogenetic control of gut bacterial metabolism to promote longevity.

Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.

Alternative Substrate Metabolism in Yarrowia lipolytica.

Recent advances in genetic engineering capabilities have enabled the development of oleochemical producing strains of Yarrowia lipolytica. Much of the metabolic engineering effort has focused on pathway engineering of the product using glucose as the feedstock; however, alternative substrates, including various other hexose and pentose sugars, glycerol, lipids, acetate, and less-refined carbon feedstocks, have not received the same attention. In this review, we discuss recent work leading to better utilization of alternative substrates. This review aims to provide a comprehensive understanding of the current state of knowledge for alternative substrate utilization, suggest potential pathways identified through homology in the absence of prior characterization, discuss recent work that either identifies, endogenous or cryptic metabolism, and describe metabolic engineering to improve alternative substrate utilization. Finally, we describe the critical questions and challenges that remain for engineering Y. lipolytica for better alternative substrate utilization.

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway.

BACKGROUND: The oleaginous yeast, Yarrowia lipolytica, has been utilized as an industrial host for about 60 years for various applications. Recently, the metabolic engineering of this host has become increasingly popular due to its ability to accumulate lipids as well as improvements made toward developing new genetic tools. Y. lipolytica can robustly metabolize glucose, glycerol, and even different lipid classes. However, little is known about its xylose metabolizing capability. Given the desirability of having a robust xylose utilizing strain of Y. lipolytica, we performed a comprehensive investigation and elucidation of the existing components of its xylose metabolic pathway. RESULTS: A quick and efficient means of determining functionality of the candidate xylose pathway genes (XYR, XDH, and XKS) from Y. lipolytica was desirable. We challenged Escherichia coli mutants lacking either the xylose isomerase (xylA) gene or the xylulose kinase (xylB) gene to grow on xylose minimal media by expressing the candidate genes from Y. lipolytica. We showed that the XKS of Y. lipolytica is able to rescue xylose growth of E. coli ΔxylB, and the XDH enabled growth on xylitol, but not on xylose, of E. coli ΔxylA. Overexpression of XKS and XDH in Y. lipolytica improved growth on xylitol, indicating that expression of the native enzymes was limiting. Overexpression of XKS and XDH in Y. lipolytica also enables robust growth on xylose under high nitrogen conditions without the need for adaptation. These results prove that a complete xylose pathway exists in Y. lipolytica, but the pathway is poorly expressed. To elucidate the XYR gene, we applied the E. coli ΔxylA xylose growth challenge with 14 candidate XYR genes and XDH. The XYR2 candidate was able to rescue growth of E. coli ΔxylA xylose on minimal media. CONCLUSIONS: While a native xylose pathway exists in Y. lipolytica, the microorganism's inability to grow robustly on xylose is an effect of cryptic genetic circuits that control expression of key enzymes in the metabolic pathway. We have characterized the key enzymes associated with xylose metabolism and demonstrated that gene regulatory issues can be overcome using strong hybrid promoters to attain robust growth on xylose without adaptation.