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    Beatrice Bishop Berle Award

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    The Beatrice Bishop Berle Award was presented to Dr. Vincent P. Dole in 1995 by the Albert Einstein College of Medicine, Division of Substance Abuse A gift from Mary Lee Gupta Beatrice Bishop Berle (1902-1993), was an American physician, teacher and author. Dr. Berle, who ran a neighborhood health clinic in East Harlem from 1953 until 1962, took a pioneering approach to family medicine by treating the entire family for the effects of heroin abuse by a member. She also helped to establish methadone maintenance as a significant treatment for heroin abuse. Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1041/thumbnail.jp

    Inter-Organellar Nucleic Acid Communication by a Mitochondrial tRNA Regulates Nuclear Transcription

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    Balancing metabolic demands with transcriptional output requires efficient communication between mitochondria and the nucleus. However, the mitochondrial factors that mediate signals to the nucleus remain poorly defined. In eukaryotes, the mitochondrial genome encodes transfer RNAs (mito-tRNAs) that function in mitochondrial-specific translation. Here, we report the detection of multiple mito-tRNAs within the nucleus of human cells. Focused studies of one such nuclear-transported mito-tRNA-asparagine (mito-tRNA-Asn) revealed that its cognate charging enzyme (NARS2) is also present in the nucleus. Nuclear localization of mito-tRNA-Asn and NARS2 was dependent on the VDAC1 mitochondrial channel and importin-α nuclear transport factor, respectively. Mito-tRNA-Asn promoted the interaction of NARS2 with histone deacetylase 2 (HDAC2) and repressed HDAC2 association with chromatin. Accordingly, inhibiting tRNA-dependent NARS2-HDAC2 complex formation licensed HDAC2 binding to target gene loci and elicited transcriptional silencing. Interfering with the mito-tRNA-Asn/NARS2/HDAC2 axis elicited metabolomic alterations in glycolytic and TCA intermediates, amino acids, and nucleotide biosynthesis. Importantly, mitotRNA- dependent transcriptional repression of glutaminase diverted glutamine towards the synthesis and maintenance of nucleotide pools, and enhanced cancer cell growth. These findings uncover nucleic-acid mediated communication between two organelles and the existence of a machinery for nuclear gene control by a mito-tRNA that restricts growth through metabolic control

    On the High-Dimensional Geometry of Neuronal Population Dynamics

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    The brain’s remarkable computational properties arise from the collective activity of up to billions of densely interconnected neurons. Neurotechnologies to simultaneously measure the activity of many neurons have been steadily developed over the past decades, with mesoscopic optical imaging now reaching tens of thousands or more neurons in a single experiment. These data have only opened up additional fundamental questions on how the neuronal population code enables robust yet flexible computation from the highly variable activities of single neurons. Here, we utilized large-scale optical imaging to investigate how neuronal population dynamics are structured across diverse brain regions during spontaneous and variable behaviors. In the first part, we investigated the dimensionality and spatiotemporal structure of cortex-wide dynamics during spontaneous and uninstructed behaviors in the mouse, encompassing up to one million neurons recorded simultaneously and at multi-Hertz volume rates. While more than a decade of work has suggested that neuronal population dynamics appear to lie on low-dimensional manifolds that capture a large degree of neural variance and other sensorimotor features, recent evidence has suggested that ongoing dynamics in the brain exhibit higher dimensionality than previously appreciated. We found that the measured dimensionality of neuronal dynamics is even more high-dimensional than previously shown, and that the measured dimensionality scaled in an unbounded fashion with the number of recorded neurons. Within these dimensions, covarying ensembles of neurons were highly distributed across the entire dorsal cortex and relatively few were related to spontaneous behavior, suggesting that the majority of identified neural dimensions uniquely captured by large-scale recording were related to purely internal processing. Next, we switched our focus to the larval zebrafish in order to ask how highdimensional whole-brain dynamics produce population codes that are robust yet flexible enough to generate variable behaviors. To do so, we honed in on a regime of visual object size — between those that elicit hunting and avoidance behaviors — which induced maximum behavioral variability. We found that the visual encoding of object size is robust at the population level, despite the highly variable activity of single neurons. This robustness despite variability was due to the multi-dimensional geometry of the neuronal population dynamics: trial-to-trial “noise” modes were largely orthogonal to sensory encoding dimensions. Finally, we showed that these many of these noise modes were actually related to the larva’s behavior. Within this variability, we identified two brain-wide neuronal populations whose pre-motor activity predicted whether the larva would respond to a stimulus and, if so, which direction it would turn on a single-trial level. These populations were able to predict such single-trial behavior even seconds before the stimulus onset, suggesting they encoded time-varying internal biases that modulated the larva’s behavior, perhaps organizing behavior over longer timescales. In both the mouse and larval zebrafish, we found that neuronal population dynamics were extremely high-dimensional; mixed at the single neuron level, which can initially appear as “noisy” variability, but robust and highly structured at the population level; spatiotemporally structured in covarying ensembles that are distributed brain-wide; and dominated by the encoding of motor behavior and behaviorallyrelevant information, even in “non-motor” areas such as primary sensory regions

    A Chemical-Genetic Map of Drug Resistance in Mycobacterium Tuberculosis

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    Tuberculosis (TB) is the leading cause of death from any single infectious disease, killing over 1.5 million people each year. Treatment of TB is extraordinarily difficult, requiring at least 6 months of combination antibiotic therapy to cure patients. Even with this prolonged treatment, 5 to 10% of patients are not fully cured and experience disease relapse. Mycobacterium tuberculosis (Mtb), the causative agent of TB, is intrinsically resistant to most antibiotics and can acquire an additional level of antibiotic resistance through specific chromosomal mutations. A better understanding of intrinsic and acquired drug resistance in Mtb is essential to develop faster treatments and better drug resistance diagnostics. We sought to understand the genetic basis drug resistance in Mtb using a novel CRISPR interference (CRISPRi)-based chemical genetics platform. Our lab developed a CRISPRi library containing 100,000 unique knockdown strains and treated this library with a panel of some of the most important antitubercular drugs. The fitness of each mutant in the library was assessed by next generation sequencing. Across over thirty different drug treatment conditions, we identified hundreds of chemical-genetic hits. We were then able to leverage this data set to gain novel biological insights into the mechanisms of intrinsic drug resistance in Mtb and identify genetic strategies to disarm these mechanisms to potentiate the activity of existing antibiotics. These data provided a nuanced picture of how the mycobacterial cell envelope serves as a selective barrier to antibiotic penetration. We found that antibiotic activity can be potentiated via selective inhibition of cell envelope synthetic enzymes as well as regulatory proteins. These data also led to the identification of a novel ribosomal protection protein, which we have termed OcrA, that confers resistance against ribosome inhibitors such as linezolid and chloramphenicol. This may allow for the rational engineering of linezolid analogs to avoid the activity of OcrA. The CRISPRi chemical-genetic screen data was also used to uncover several novel mechanisms of acquired drug resistance amongst clinical Mtb isolates. Among the strongest hits identified from these chemical-genetic screens was ettA, a ribosome-associated ATPase that regulates translation initiation. CRISPRi mutants for ettA were resistant to a diverse set of drugs including streptomycin, levofloxacin, and ethambutol. Mining a whole genome sequence database of over 40,000 clinical Mtb isolates we identified several ettA mutations which phenocopy the CRISPRi mutants and confer multidrug resistance. Of particular interest, strains harboring an EttA ATP binding site mutation (Gly41Glu) were found to be concentrated in South America, especially in Perú, the location of a widespread multidrug-resistant TB outbreak. Molecular epidemiology suggests that this mutation likely arose early in the TB chemotherapy era. By conferring low-level resistance to multiple antibiotics, it likely served as a stepping-stone to the evolution of higher order drug resistance mutations. A current collaborative project is aimed at investigating the role of this ettA mutation in patient treatment outcomes in Perú

    Nathan B. Eddy Memorial Award

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    The Nathan B. Eddy Memorial Award was given to Dr. Vincent P. Dole in 1982 A gift from Mary Lee Gupta The Nathan B. Eddy Memorial Award was established in memory of one of the pioneers in the field of drug dependence following his death in 1973. The award acknowledges outstanding research efforts that have advanced our knowledge of drug dependence. Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1038/thumbnail.jp

    Details of the Exhibit

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    Description Details of the exhibit E.G.D. Cohen: A Leader in Statistical Physics Idea, design - Olga Nilova, Special Collections Librarian Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/cohen-leader-in-statistical-physics/1011/thumbnail.jp

    New York State Office of Alcohol and Substance Abuse Service Proclamation

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    New York State Office of Alcohol and Substance Abuse Service Proclamation, 2001 A gift from Mary Lee Gupta Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1045/thumbnail.jp

    Using Chemical Probes to Examine Cellular Activities

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    Chemical probes are valuable tools for investigating rapid cellular processes, such as anaphase in cell division, by allowing for the modulation of protein activity over the course of minutes. In addition, resistance to chemical probes by mutation can provide insight into inhibitor binding interactions and may provide morphological pharmacodynamic markers that allow for the identification of drug resistance or sensitivity. Here, I present a two-part thesis in which I (i) explore the role of spastin during late anaphase and nuclear envelope reformation using our recently developed spastin inhibitor and (ii) use inhibitor resistance to identify morphological markers of drug resistance via high-content microscopy. In the first part of my thesis, I use chemical probes to examine spastin’s role in cell division. Spastin is a AAA (ATPases associated with diverse cellular activities) protein that is recruited in anaphase by the endosomal sorting complex required for transport (ESCRT) to the reforming nuclear envelope, where spastin is proposed to sever stable microtubules. However, we currently lack an understanding of spastin’s dynamics during anaphase, particularly the roles of microtubules and ESCRT proteins in said dynamics. Using live cell imaging and chemical probes that inhibited spastin’s ATPase activity (spastazoline) or affected microtubules (nocodazole, taxol, or monastrol), I quantified spastin dynamics during anaphase. Spastin foci accumulated on the periphery of chromosomes and were similar on the spindle pole-facing and midzone-facing sides of chromosomes. However, foci that colocalized with microtubules persisted longer than microtubule-free foci. Inhibiting spastin with spastazoline resulted in more stable microtubule-proximal foci compared to microtubule-free foci. Spastazoline had no measurable effect on the accumulation of the ESCRT-III protein, charged multivesicular body protein 4B (CHMP4B). Together, these data suggest that spastin dynamics during late anaphase are decoupled from ESCRT dynamics and are instead modulated by the presence of microtubules and spastin’s ATPase activity. In the second part of my thesis, I use chemical probes to examine morphological markers of drug resistance. Drug resistance is a confounding factor in the treatment of many diseases, particularly cancers, where uncontrolled and error-prone cell divisions can give rise to resistance-conferring mutations. While identifying drug-resistant cell populations in advance could help personalize therapeutics and prevent delays in administering effective treatments, we currently lack rapid methods for broadly analyzing drug resistance. Here, I help generate drug-resistant cell lines and image these cells using Cell Painting, a high-content microscopy method, to characterize morphological markers of resistance. Working in collaboration with colleagues at the Broad Institute, we found that our data support the identification of resistant cell lines using profiles generated from high-content microscopy. This work suggests that image-based cell profiling may be a valuable method of identifying drug-resistant or -sensitive cells and could be a useful tool to guide therapeutic strategies

    Life on a Ball: Multi-Week Tethered Behavior in Drosophila

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    By studying head fixed or rigidly tethered animals, neuroscientists have been able to make high-resolution neurophysiological and behavioral measurements during naturalistic tasks. Tethered paradigms have a particularly rich history in the study of insect behavior and in this thesis I describe technology that extends by one-hundred fold|from hours to weeks|the timescale over which individual Drosophila can be routinely studied while tethered in place

    Albert Einstein Award

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    Dr. Vincent P. Dole\u27s Albert Einstein Award For Excellence in Psychiatry and Related Disciplines A gift from Mary Lee Guptahttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1037/thumbnail.jp

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