Scalable CRISPR Screens in Zebrafish Using MIC-Drop.

CRISPR-Cas9 is a powerful tool to interrogate gene function in a targeted and systematic manner. Although the technology has been scaled up for large-scale genetic screens in cell culture, similar scale screens in vivo have been extremely challenging due to the cost, labor, and time required to generate and keep track of thousands of mutant animals. We reported the development of Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform that makes large-scale reverse genetic screens possible in zebrafish.

A zebrafish model of combined saposin deficiency identifies acid sphingomyelinase as a potential therapeutic target.

Sphingolipidoses are a subcategory of lysosomal storage diseases (LSDs) caused by mutations in enzymes of the sphingolipid catabolic pathway. Like many LSDs, neurological involvement in sphingolipidoses leads to early mortality with limited treatment options. Given the role of myelin loss as a major contributor toward LSD-associated neurodegeneration, we investigated the pathways contributing to demyelination in a CRISPR-Cas9-generated zebrafish model of combined saposin (psap) deficiency.

Top2a promotes the development of social behavior via PRC2 and H3K27me3.

Little is understood about the embryonic development of sociality. We screened 1120 known drugs and found that embryonic inhibition of topoisomerase IIα (Top2a) resulted in lasting social deficits in zebrafish. In mice, prenatal Top2 inhibition caused defects in social interaction and communication, which are behaviors that relate to core symptoms of autism.

The 5α-reductase inhibitor finasteride reduces opioid self-administration in animal models of opioid use disorder.

Opioid use disorder (OUD) has become a leading cause of death in the United States, yet current therapeutic strategies remain highly inadequate. To identify potential treatments for OUD, we screened a targeted selection of over 100 drugs using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior.

Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish.

Extensive characterisations of the zebrafish genome and proteome have established a foundation for the use of the zebrafish as a model organism; however, characterisation of the zebrafish lipidome has not been as comprehensive. In an effort to expand current knowledge of the zebrafish sphingolipidome, a Parallel Reaction Monitoring (PRM)-based liquid chromatography-mass spectrometry (LC-MS) method was developed to comprehensively quantify zebrafish ceramides. Comparison between zebrafish and a human cell line demonstrated remarkable overlap in ceramide composition, but also revealed a surprising lack of most sphingadiene-containing ceramides in the zebrafish.

Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence.

A LC-MS-based workflow for measurement of branched fatty acid esters of hydroxy fatty acids.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous mammalian lipids with antidiabetic and anti-inflammatory effects. We previously identified 16 different FAHFA families, such as branched palmitic acid esters of hydroxy stearic acids (PAHSAs); each family consists of multiple isomers in which the branched ester is at different positions (e.g.

Regulation of mitochondrial ceramide distribution by members of the BCL-2 family.

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging.

A nonapoptotic role for BAX and BAK in eicosanoid metabolism.

BCL-2 proteins are key regulators of programmed cell death. The interplay between pro and antiapoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key nonapoptotic roles for several BCL-2 proteins.

Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects.

Increased adipose tissue lipogenesis is associated with enhanced insulin sensitivity. Mice overexpressing the Glut4 glucose transporter in adipocytes have elevated lipogenesis and increased glucose tolerance despite being obese with elevated circulating fatty acids. Lipidomic analysis of adipose tissue revealed the existence of branched fatty acid esters of hydroxy fatty acids (FAHFAs) that were elevated 16- to 18-fold in these mice.

Molecular network analysis of phosphotyrosine and lipid metabolism in hepatic PTP1b deletion mice.

Metabolic syndrome describes a set of obesity-related disorders that increase diabetes, cardiovascular, and mortality risk. Studies of liver-specific protein-tyrosine phosphatase 1b (PTP1b) deletion mice (L-PTP1b(-/-)) suggest that hepatic PTP1b inhibition would mitigate metabolic-syndrome through amelioration of hepatic insulin resistance, endoplasmic-reticulum stress, and whole-body lipid metabolism. However, the altered molecular-network states underlying these phenotypes are poorly understood.

Emerging roles of lipids in BCL-2 family-regulated apoptosis.

Apoptosis is an intricately regulated process required for the health and homeostasis of living systems. The mitochondrial apoptotic pathway depends on the BCL-2 family of pro- and anti-apoptotic members whose interactions form a complex network of checks and balances in regulating cell fate. A diverse set of signals recruits distinct BH3-domain only BCL-2 proteins to trigger activation of the executioner proteins BAX and BAK.

The 2.5 Å structure of CD1c in complex with a mycobacterial lipid reveals an open groove ideally suited for diverse antigen presentation.

CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-β1-phosphomycoketide (MPM).

Reversible switching of the shear modulus of photoresponsive liquid-crystalline polymers.

Manipulation makes light work: The morphology and rheological properties of a liquid-crystalline system can be dynamically manipulated with UV light by attaching photoresponsive liquid-crystalline moieties to a siloxane-based polymer. Stimulation with UV light induces a conformational change in the molecule, which disrupts the liquid-crystalline mesophase (see picture), and results in a dramatic change in its rheological properties.

Controlling the morphology of side chain liquid crystalline block copolymer thin films through variations in liquid crystalline content.

In this paper, we describe methods for manipulating the morphology of side-chain liquid crystalline block copolymers through variations in the liquid crystalline content. By systematically controlling the covalent attachment of side chain liquid crystals to a block copolymer (BCP) backbone, the morphology of both the liquid crystalline (LC) mesophase and the phase-segregated BCP microstructures can be precisely manipulated. Increases in LC functionalization lead to stronger preferences for the anchoring of the LC mesophase relative to the substrate and the intermaterial dividing surface.

Influence of variations in liquid-crystalline content upon the self-assembly behavior of -based block copolymers.

A series of well-defined smectic side chain liquid-crystalline (LC) block copolymers with a low glass transition (Tg) siloxane block has been synthesized via anionic polymerization; these systems consist of a glassy polystyrene block and a unique low glass transition temperature LC block based on poly(vinylmethylsiloxane) to which six different LCs have been synthesized and attached. The synthesis techniques used provide systematic control over covalent LC side chain content, allowing for a range of morphologies to be obtained from a single block copolymer backbone during a one-step LC attachment reaction. Variations in the LC structure and content significantly affect the morphology of the LC mesophase, allowing the smectic-to-isotropic transition temperature to be tuned from room temperature up to 150 °C.