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Transgenic Tools in Ants and the Representation of Alarm Pheromones in the Ant Antennial Lobe
Full text linkFor decades, ants have served as major study species for ethologists, theorists, geneticists, and chemical ecologists, who have been drawn to understand how their unique features contribute to the evolution and maintenance of insect societies. These features, especially their extreme morphological plasticity, collective behavior, and complex chemical communication, together with their small sizes and relatively simple brains, make ants intriguing model systems for many topics in neurobiology. While many ant species possess brains no larger than the wellcharacterized vinegar fly, the primary olfactory processing centers (antennal lobes) contain an order of magnitude more functional units compared to the vinegar fly, presumably to facilitate detecting and discriminating between vast numbers of pheromones. The discovery of additional developmental differences between ants and flies led to a proposed but untested model where ant olfactory sensory neurons target the appropriate antennal lobe compartment via receptor dependent activity, as occurs in mammals. However, while transgenic tools have allowed dissection of the olfactory system\u27s development and functional organization in some solitary insect species, these tools have so far been impossible to implement in ants. Taking advantage of the unusual experimental tractability of the clonal raider ant Ooceraea biroi, we implemented piggyBac transgenesis for the first time in ants, and generated a toolkit of transgenic lines. These protocols and transgenic tools greatly expand the space of feasible experiments in ants, and make clonal raider ants the most experimentally tractable model system among eusocial insects. We then used these transgenic lines to study outstanding questions in social insect olfaction
River Campus Promenade
No full textRiver Campus promenade (looking south), 2023
Photo by Juan Rodriguezhttps://digitalcommons.rockefeller.edu/river_campus/1073/thumbnail.jp
Hypodermic Syringe in Metal Case
No full textHypodermic syringe, steel and glass in metal case, ca. 1930s
Donated by the RU Hospital
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1043/thumbnail.jp
Deep Molecular Characterization of Human Cortical Cell-Types
Full text linkHuman cortical cytoarchitecture is as beautiful as it is complex. Harboring dozens of morphologically, functionally, and molecularly distinct cell-types, the neocortex is organized into cytoarchitectonically and functionally diverse regions. In concert, the diverse cell-types that are found within the diverse cortical regions build the center of all higher cognitive functions, including but not limited to sensory processing, instruction and execution of motor movements, abstract thinking, perception, as well as speech processing and production. Over the past decades, detailed knowledge on the molecular characteristics of cortical cell-types has been obtained from genetically engineered animal models. These deep insights into the molecular underpinnings of cortical cell-types, paired with recent advances in cellular nuclei isolation technologies allow for the isolation and deep molecular profiling of human neocortical cell-types. We developed a novel serial fluorescence activated nuclei sorting strategy, which we will henceforth refer to as sFANS. sFANS stands for serial fluorescence activated nuclei sorting and allows for the isolation of up to sixteen cell-types from the human cerebral cortex. In this thesis, we present data from fourteen routinely isolated cell-types, which include layer 2/3, layer 4, region specific populations of layer 5, termed layer 5 region-specific (layer 5rs), layer 5a, as well as layer 6a, and layer 6b excitatory pyramidal neurons, VIP, LAMP5, RELN, and PVALB expressing interneurons, astrocytes, microglia, oligodendrocytes, and OPCs. We show that our isolation strategy is highly cell-type specific and reproducible, permitting deep molecular profiling of the cell-types across several regions of post-mortem human cortex. Moreover, we show that isolated and cell-type specific populations can be passed on to a number of different downstream assays. Specifically, in the course of this work, we conducted RNAseq, snRNAseq, ATACseq, as well as CAG expansion assays on hundreds of cell-type specific populations and these data, obtained from several different regions of the human neocortex, will be presented in this work. As many neurological diseases are known to affect the neocortex, cell-type specific studies are instrumental for our understanding of the underlying pathophysiological and molecular alterations that underly these conditions. It has been widely recognized that cell-type specific knowledge is critically needed to bring us closer to more effective treatment strategies. Here, we focused our efforts on the investigations of the neuronal neocortical alterations observed in Huntington\u27s disease. Our precise and reproducible methodology revealed that an HTR2C expressing layer 5a excitatory neuron population is selectively vulnerable and drastically diminished across all stages of Huntington\u27s disease. In collaborative studies, in which we used samples from an AAV2.retro, striatal injected nonhuman primate model, we show that the vulnerable HTR2C expressing neurons constitute a cortico-striatal projecting cell-type. Moreover, we quantified the relative number of nuclei per cell-type using a population enriched snRNAseq strategy and, while our cell-type specific CAG expansion assays confirmed dramatic expansions in all deep layer excitatory neurons, only those nuclei of the layer 5a type were diminished in Huntington\u27s disease. Our data therefore suggest that CAG expansion is necessary but not sufficient to drive the loss of HTR2C expressing layer 5a excitatory neurons and that potential alterations of the cortico-striatal connections that are formed by this particular cell-type may be a major driver for the vulnerability of HTR2C expressing neurons in Huntington\u27s disease. Chapter one of this thesis offers an introduction into the neuroanatomical aspects of the human neocortex. The major cell-types that constitute the cortex, their morphological, functional, and molecular hallmarks will be introduced. Functionally and cytoarchitectonically diverse regions of cortex that are of particular importance to this work will be discussed and several aspects of regional and cellular connectivity will be highlighted. Chapter two provides background and rationale for the development of our sFANSeq isolation strategy. Detailed information on our methods, including nuclei isolation, labeling, and gating strategies are provided. Results and confirmation of reproducibility and cell-type specificity will be presented. In chapter three, we introduce current knowledge on the neuropathological alterations of Huntington\u27s disease with a focus on the neocortex and expand on this knowledge through presentation and discussion of the results and findings from our sFANS experiments, RNAseq, snRNAseq, ATAC, and CAG expansion data. This work is intended to provide the ground stone for the application of our sFANS strategy for the isolation and deep characterization of cortical cell-types in human health and disease. Our aim is to introduce the sFANS strategy as a tool that provides highly robust, reproducible, and celltype specific results for all major human neocortical cell-types
Kravis Research Building
No full textKravis Research Building, lobby
Photo by Juan Rodriguez
The Marie Josee and Henry R. Kravis Research Building can accommodate over 600 scientific personnel. The large floor plans are designed to encourage collaboration between scientists, and by eliminating the interior structural columns and mechanical shafts found in most older buildings, the laboratory layouts may be modified to serve future research requirements.https://digitalcommons.rockefeller.edu/river_campus/1076/thumbnail.jp
Bioorthogonal Tethering of Drug Fragments to Engineered G Protein-Coupled Receptors
Full text linkG protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures for nearly 100 discrete GPCRs in complex with various ligands, discovering allosteric drugs that can modulate receptor signaling remains challenging. In principle, allosteric ligands have certain advantages because they bind to pockets on the receptor that do not directly impact the orthosteric site for agonist ligands, but still tune downstream signaling pathways. But identifying and validating allosteric ligands remains difficult partly due to the dynamic nature of GPCRs in native membranes. Of the more than 3700 distinct FDA-approved drugs, there are only 19 that are allosteric, and only seven target GPCRs. Many early-stage allosteric modulators fail in the latestages of the drug development pipeline because they are designed based on static structures that fail to recapitulate biological complexity. New methodologies to probe functional relevance of allosteric lead compounds in live-cell systems early in drug discovery are needed. However, the dynamic nature of allosteric sites and low-affinities of starting compounds at concentrations amenable to cell-based screening poses a significant challenge for probing protein allosteric sites. This thesis presents a novel strategy employing genetic code expansion (GCE) and bioorthogonal chemical reactions for tethering drug fragments adjacent to allosteric sites in GPCRs to increase their potency and enable fragment-based drug screening in cellular systems. C-C chemokine receptor 5 (CCR5) was selected as a model receptor for proof-of-concept studies. First, we developed a luciferase-based reporter assay to compare and optimize side-byside the efficiency of incorporation of three noncanonical amino acids (ncAAs) at three sites on CCR5 using three distinct genetic code expansion plasmid systems. Satisfactory incorporation of each of the tested ncAAs into heterologously-expressed CCR5 was achieved. Cell-based calcium mobilization assays were carried out to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered variants using heterobifunctional compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Bioorthogonal labeling of CCR5 in live cells using inverse electron demand Diels-Alder ligation was more specific and efficient than the strain promoted azide-alkyne cycloaddition reaction assessed. GCE was then employed to site-specifically introduce one of three different reactive ncAAs in CCR5 near an allosteric binding site for the drug maraviroc (mvc). Molecular dynamics simulations were used to design heterobifunctional mvc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction with the ncAAs. We synthesized a library of these analogues and employed the bioorthogonal tethering reactions to couple the analogues to the engineered CCR5 in live cells, which were assayed using cell-based signaling assays. Tetherable low-affinity mvc fragments had higher potency for CCR5 engineered with reactive ncAAs that were adjacent to the mvc binding site. The strategy we describe to tether drug fragments to GPCRs should prove useful in probing allosteric or cryptic binding sites in fragment-based GPCR-targeted drug discovery
Lauritsen Electroscope; Details
No full textLauritsen electroscope, accession no.41; circa 1940
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/scientific-instruments/1022/thumbnail.jp
Janice Carissa, Piano
No full textLast concert
Janice Carissa, piano, performed Bach-Busoni: Toccata and Fugue in D Minor; Granados: Allegro de Concierto; Rzewski: Winnsboro Cotton Mill Blues; Scriabin: Sonata No. 3
Photo by Sienny Deborahttps://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1011/thumbnail.jp
Danny Driver, Piano
No full textPost-pandemic Season Opening Concert.
Danny Driver, piano, performed Bach: French Suite #5 in G Major; Ligeti: Arc-en-ciel – Autumn in Warsaw, Fanfares; Schumann: Symphonic Etudes, Op.13 (1852 version)
Photo Copyright © Danny Driverhttps://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1008/thumbnail.jp
New Insights into the Roles of FAN1 Nuclease in Genome Maintenance and Disease
No full textExogenous and endogenous insults to genomic integrity are an existential threat to every species, and therefore robust DNA damage repair mechanisms have evolved to protect against genotoxic threats. FANCD2 and FANCI associated nuclease 1 (FAN1) has roles in protection against two major threats to genomic stability: DNA interstrand crosslinks (ICLs), and the expansion of trinucleotide repeats. ICLs are highly deleterious lesions that disrupt replication, are destructive to dividing cells, and are extremely toxic to the hematopoietic system. The replication-dependent Fanconi anemia (FA) pathway defends against ICL toxicity in rapidly dividing cells. However, while it is critical for protecting the bone marrow, the FA pathway is ineffective in the less-proliferative tissues of the kidney and liver. FAN1 interacts with FA pathway proteins and has been shown to process ICLs in biochemical studies. Depletion of FAN1 sensitizes cells to ICL-inducing agents in vitro, but does not cause bone marrow failure in vivo. Rather, it leads to a hereditable chronic kidney disease called karyomegalic interstitial nephritis (KIN), characterized by widespread karyomegaly, end stage renal disease, and liver dysfunction, suggesting FAN1 may play an important genoprotective role in the kidney and liver. Yet a complete picture of the role of FAN1 in DNA damage repair has been hampered by potential compensatory activity of other nucleases in the setting of FAN1 deficiency. The Smogorzewska laboratory has previously shown evidence of redundancy between FAN1 and the nuclease SNM1A/DCLRE1A, which has also been shown to process ICLs. Here, I describe a synergistic ICL repair deficiency phenotype in mouse embryonic fibroblasts derived from Fan1−/− Snm1a−/− (dKO) mice, which exceeds that of FA pathway-deficient cells. In vivo, dKO mice exhibit profound karyomegaly in the kidney and liver. However, the mice exhibit no spontaneous bone marrow deficiency. Even when the hematopoietic system is chronically stressed with a viral memetic the dKOs exhibit no bone marrow failure. Taken together, my results demonstrate that FAN1 and SNM1A participate together in critical genome maintenance activity where the FA-pathway is insufficient. In addition to driving KIN, variants in FAN1 have also been associated with earlier onset of Huntington\u27s disease (HD), a trinucleotide repeat expansion disease. Depletion of FAN1 increases CAG repeat expansion rate in vitro and in a mouse model of HD. FAN1 also interacts with the mismatch repair (MMR) protein MLH1, which, together with MLH3, is indispensable for CAG repeat expansion in HD. Here, I show that FAN1 interacts with MLH1 via a well conserved MLH1- interacting peptide motif (MIP-box) on FAN1, which is necessary for FAN1-MLH1 interaction. I identify other human MIP-box proteins, including EXO1 and BLM, using in silico modeling I locate the MIP-box binding site (MIS) on MLH1, and using in vitro experiments I show that mutating the MIS abrogates MLH1 interaction with FAN1, EXO1, and BLM. Using biochemical assays, I show that all three MIP-box proteins bind the MLH1-MLH3 heterodimer directly. Based on in silico models, I predict this direct interaction is between MIP-box proteins and MLH1, and that docking of a MIP-box in the MLH1 MIS does not prevent the formation of the MMR heterodimers MLH1-MLH3, MLH1-PMS2, and MLH1- PMS1. A biomathematical model of competition between MIP-box proteins for binding the MLH1 MIS suggests increased interaction between BLM and MLH1 when FAN1 or EXO1 is depleted. I propose a model in which FAN1 suppresses CAG repeat expansion by regulating the amount of BLM that can bind MLH1, which delivers BLM to CAGinduced hairpins, driving repeat expansion. Finally, I describe the experiments necessary to validate the model