Characterizing the frequency and indications for repair of abdominal aortic aneurysms with diameters smaller than recommended by societal guidelines.

OBJECTIVE: Although the Society for Vascular Surgery recommends repair of abdominal aortic aneurysms (AAA) at 5.5 cm or greater in men and 5.0 cm or greater in women, AAA repair below these thresholds has been well-documented. There are clear indications for repair other than these strict size criteria, but the expected proportion of such repairs in one's practice has not been studied. We sought to characterize the indications for repairs of aneurysms below diameter recommendations at a single academic center. Using the assumption that this real-world experience would approximate that of other practices, we then used national data to extrapolate these findings. METHODS: A single-center retrospective review was conducted of all elective open AAA (oAAA) and endovascular aneurysm repair (EVAR) from 2010 to 2020 to assess the incidence of and indications for repair of aneurysms below diameter recommendations (defined as <5.5 cm in men and <5.0 cm in women). Reasons for these repairs were defined as (1) iliac aneurysm, (2) saccular morphology, (3) rapid expansion, (4) patient anxiety, (5) distal embolization, (6) other, and (7) no documented reason. The Vascular Quality Initiative (VQI) was queried for all asymptomatic oAAA and EVAR (2010-2020) and repairs below diameter recommendations were identified. Findings from the single-center analysis were applied to the VQI cohort to extrapolate estimates of reasons for repairs done nationally. In-hospital mortality and major adverse cardiac events (MACE) were compared between those below size recommendations and those meeting size recommendations. RESULTS: Of 456 elective AAA repairs at our center, 147 (32%) were below size recommendations. This finding was more common for EVAR (35% vs 28%). Reasons were: not documented (41%), iliac aneurysm (23%), saccular (10%), rapid expansion (10%), patient anxiety (7%), other (6%), and distal embolism (3%). Of 44,820 elective AAA repairs in the VQI, 17,057 (38%) were below size recommendations (40% EVAR, 26% oAAA). Patients who were repaired below size recommendations had lower in-hospital death (oAAA, 2.4% vs 4.6% [P < .0001]; EVAR, 0.3% vs 0.8% [P < .0001]). When single-center findings were applied to the VQI dataset, an estimated 10,064 repairs were performed nationally for acceptable indications other than size criteria. Conversely, there may have been 6993 repairs (with an associated 35 deaths) performed without documented indication. CONCLUSIONS: Repairs for AAA below the recommended diameter guidelines account for approximately one-third of all elective AAA procedures in both the VQI and our single-center experience. Assuming our practice is typical, nearly 60% of repairs below size recommendations meet the criteria for other clear reasons. The remaining 40% lack a documented reason, meaning that 13% of all elective AAA repairs were done for aneurysms below size recommendations without an acceptable indication. As awareness of overuse and underuse is heightened, these data help to estimate the expected proportion of repairs for less common pathologies. They also provide a potential baseline data point for efforts at decreasing overuse.

Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.

Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.

Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells.

RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.

A regulatory network of Drosophila germline stem cell self-renewal.

Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

Conserved regulators of nucleolar size revealed by global phenotypic analyses.

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.

Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.