Renal L-2-hydroxyglutarate dehydrogenase activity promotes hypoxia tolerance and mitochondrial metabolism in Drosophila melanogaster.

Objectives: The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh.

NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA.

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout.

The RNA-binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance.

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here, we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing is critical for Drosophila melanogaster intestinal stem cells and their daughter cells, enteroblasts, to maintain their progenitor cell properties and functions.

Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity.

The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly.

I-KCKT allows dissection-free RNA profiling of adult intestinal progenitor cells.

The adult intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed (), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.

Adapting Cell Lines to Serum-Free Culture Conditions.

Successful cell culture relies on media containing xenogenic components such as fetal bovine serum to support continuous cell proliferation. Here, we report a serum-free culture condition that supports the growth and proliferation of S2R+ and Kc167 cell lines. Importantly, the gradual adaptation of S2R+ and Kc167 cells to a media lacking serum was supported by supplementing the media with adult soluble extract, commonly known as fly extract.

Identification and Characterization of Breakpoints and Mutations on Balancer Chromosomes.

Balancers are rearranged chromosomes used in to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in causes loss of radiation-induced apoptosis and a breakpoint in causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation.

Canonical nucleators are dispensable for stress granule assembly in intestinal progenitors.

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Although SGs have been extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remains poorly characterized. Here, we report that the orthologs of the mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP and TIAR are enriched in adult fly intestinal progenitor cells, where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs).

Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during larval development.

The dramatic growth that occurs during larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis.

Polycomb group (PcG) proteins and Pax6 cooperate to inhibit reprogramming of the developing eye.

How different cells and tissues commit to and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. Here, we demonstrate that Polycomb group (PcG) proteins maintain eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate.

Opposing Post-transcriptional Control of InR by FMRP and LIN-28 Adjusts Stem Cell-Based Tissue Growth.

Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. We report that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components.