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Interrogation and Manipulation of the IGG FC Glycan
Full text linkAntibody signaling is a cardinal feature of successful adaptive immune responses. The magnitude and direction of this signaling is determined by the structure of a given antibody\u27s fragment crystallizable (Fc) domain, which interfaces with cells of the immune system. Often considered a constant region, the immunoglobulin G (IgG) Fc protein backbone and conserved N-linked glycan combine to introduce structural diversity in IgG molecules that in turn, triggers divergent humoral responses. These N-glycans are variably constructed, ultimately leading to families of highly related, but non-equivalent glycoproteins known as glycoforms. Despite burgeoning interest in understanding the complexities of IgG Fc glycoforms and their functions, there is an evident scarcity of tools available to distinguish and target them. In addition, the highly conserved nature of the glycan and its presence on the B cell receptor (BCR) provokes questions of its possible role in the generation of antibody responses. In the first part of this thesis, I identify a novel class of synthetic nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. Applying this technology to Fc glycoforms defines nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. Solving the structure of a nanobody-Fc complex via x-ray crystallography reveals a unique mode of recognition of IgG glycoforms. By adapting nanobodies to standard biochemical methods, I clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their Fc glycan profile, selectively disrupt IgG-Fcγ receptor (FcγR) binding both in vitro and in vivo, and interrogate BCR glycan structure on living cells. In the second part of this thesis, I develop mouse models and biochemical tools to help define the role of the IgG Fc glycan in developing antibody responses. Preliminary studies show that following immunization, mice lacking the N-glycan acceptor residue, Asn 297, exhibit deficiencies in germinal center (GC) responses. In this model, B cells with an aglycosylated BCR participate less frequently in the GC reaction, bind antigen less avidly, and show signaling deficits downstream of BCR engagement. Development of an aglycosylated mouse IgG1-specific nanobody shows that in allelic competition experiments, B cells with glycosylated BCRs are favored. To aid future studies, an additional mouse model null for endogenous Fc-FcγR binding is generated. Finally, a mouse expressing human IgG1 in the heavy chain locus proves useful for chronic administration of human antibodies
Nathan B. Eddy Memorial Award
No full textThe 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
Haven Emerson Award
No full textHaven Emerson Award was given to Dr. Vincent P. Dole in 1969 for distinguished service to Public Health
A gift from Mary Lee Gupta
John Haven Emerson (February 5, 1906 – February 4, 1997) was an American inventor of biomedical devices, specializing in respiratory equipment. He is best remembered for his work in improving the iron lung.
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1040/thumbnail.jp
Meinecke & Co. Hot Water Medicine Bag
No full textMeinecke & Co. Hot Water Medicine Bag, ca. 1930s
Donated by the RU Hospital
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1044/thumbnail.jp
New York State Office of Alcohol and Substance Abuse Service Proclamation
No full textNew 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
Life on a Ball: Multi-Week Tethered Behavior in Drosophila
No full textBy 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. Specifically, I designed and constructed a new experimental rig that allows a rigidly tethered fly to live for at least two weeks within a virtual reality environment. The fly stands (and is free to walk) on an airsupported ball while viewing a virtual visual environment on a panoramic display. To enable long-term survival, the fly is fed small, nutritive drops, thousands of times over two weeks, at experimentally defined moments. The rigs are compact, such that two dozen or more can fit in a small room, allowing for the study of many flies simultaneously. The rigs are also modular, and easily adapted to new kinds of experiments. I have used them to probe innate behaviors, like sleep and navigation, over long time scales. Moving forward, they may allow scientists to train Drosophila in complex, learned, tasks because they enable the delivery of thousands of rewards over multiple weeks, which can be used as reinforcers in operant or classical conditioning paradigms. Beyond the technology, this thesis also describes a new behavioral tendency in Drosophila. Specifically, I show that when Drosophila are allowed to freely navigate in a simple virtual environment for one to two weeks, they maintain a surprisingly consistent navigational angle as they walk forward. Each fly walks along a unique, preferred, travel direction, for tens to hundreds of meters from a start location. The preferred travel direction can be considered a navigational goal angle because individuals will repeatedly correct for experimentally induced virtual rotations away from this angle. Flies require a visual orienting cue to successfully progress forward along the goal angle—walking in circles without it—and they will return to the same goal angle in the morning after spending a full night (twelve hours) without the cue. These experiments demonstrate that Drosophila can commit to a navigational goal over multiple weeks and they further argue for the existence of navigation-related signals and mechanisms in the fly brain, yet to be discovered, with similarly long persistence times
The Internal Orchestration of a Courtship Ritual in Drosophila
No full textThe diversity of behaviors exhibited by animals is astounding. Even within a species, members constantly select between a myriad of ways to interact with their environment, ranging from foraging to fighting. Yet, why an individual behaves as it does in each moment of time typically remains nebulous to us as observers. In this thesis, I explore the possibility that these seemingly idiosyncratic behavioral decisions can be understood by accessing the hidden internal motivations that spur an animal to do one thing in lieu of another. By leveraging the compact brain architecture and rich social behaviors of the fruit fly Drosophila melanogaster, I detail how internal drives are encoded in specific neural circuits, and how they pattern an animal\u27s behavior on a moment-tomoment basis by tuning both perception and action selection. In Drosophila, male flies perform a lengthy and elaborate courtship ritual that typically commences after they sample the pheromones of a suitable mate. The initiation of courtship represents a striking switch in a male\u27s behavior, as he transitions from being apathetic or blind to a female to vigorously tracking her for minutes at a time while singing a species-specific song. In chapters 2-4, I describe how the link between perception and action is dynamically reconfigured in the male brain at the onset of courtship to convert a female from an indifferent visual object to a target of desire. These studies were largely enabled by a preparation in which tethered flies can interact with a fly-sized visual target in virtual reality, which has allowed me to coordinately examine social behaviors and their underlying neural substrate for the first time. In Chapter 5, I explore how the behavioral flexibility afforded by the neural mechanisms described in the preceding chapters is employed in the context of the natural ecology of Drosophila. In the wild, flies meet and mate on fermenting fruits where a diversity of individuals congregate. Reproductive success in this complex social environment requires that a male fly not only doggedly pursues his mate of choice, but that he also effectively fights off rival males vying for the same female. Thus, a male\u27s sexual arousal must be balanced with competing drives to appropriately pattern social interactions in these naturalistic contexts. I will show that when a male transiently senses a cue emitted by male competitors, he switches from courting a visual target to exhibiting aggression towards it. These rapid behavioral transitions are mediated by a small population of higher-order neurons that function to regulate the expression of this aggression rather than courtship. Based on these results, I propose that the neural pathways mediating social interactions in the fly thus function like a switchboard, allowing incoming visual information to be flexibly rerouted to different motor pathways depending on a male\u27s motivational state and immediate sensory context to pattern his ongoing behavior. This provides a conserved logic for how the incredible diversity of behaviors expressed across the lifetime of an animal could be achieved, and suggests that we can account for much of the idiosyncrasies of animal behavior by examining ongoing transitions between competing needs and drives
Marie Nyswander Award
No full textThe First Marie Nyswander Humanitarian Award was presented to Dr. Vincent P. Dole in 2000 for the extraordinary efforts to enhance the compassionate care of addiction and pain
A gift from Mary Lee Gupta
Marie Nyswander (March 13, 1919 – April 20, 1986) was an American psychiatrist and psychoanalyst known for developing and popularizing the use of methadone to treat heroin addiction.
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1042/thumbnail.jp
A Chemical-Genetic Map of Drug Resistance in Mycobacterium Tuberculosis
No full textTuberculosis (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
Development of Improved Genetic Methods for Enterococcus Faecium
No full textEnterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient genetic methods. Current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. Additional genetic infrastructure, including robust inducible promoters and sitespecific integration vectors have yet to be developed in E. faecium. Furthermore, a programmable gene silencing tool useful to selectively perturb essential genes in bacteria remains undeveloped for E. faecium. To address these limitations in gene editing of E. faecium, Chapter 2 reports the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Here, I utilize a recombineering system using a latent bacterial prophage RecT that is compatible with E. faecium and other related species. By combining recombineering with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 counterselection, I was able to produce both scar-less mutants via point mutations as well as controllable deletions of various sizes. Additionally, I report my system to have gene editing activity with PCR generated dsDNA templates. Overall, I show the versatility of my RecT-mediated recombineering method to produce substitution, deletion, and insertion mutants in E. faecium to enable facile genetic-based studies. Notably, the expression of RecT in combination with CRISPR-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less singlestranded DNA recombineering to generate specific gene editing mutants in E. faecium. Moreover, I demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, in Chapter 3, I use CRISPR-Cas9 or dsDNA mediated recombineering to perform further knock out studies to investigate E. faecium microbiology or E. faecium host-microbe interactions. Using CRISPR-Cas9 mediated recombineering, I knocked out both genomically encoded sortase A genes in E. faecium for downstream functional characterization. Additionally, I used CRISPR-Cas9 mediated recombineering was to knock out E. faecium tyrosine decarboxylase to genetically validate E. faecium\u27s interaction with the orphan human G-protein-coupled receptor, GPRC5A. Furthermore, using dsDNA recombineering, I generated HA-tagged and knockouts of E. faecium bile acid salt hydrolase to genetically confirm E. faecium\u27s interactions with bile acids. The general RecT-mediated recombineering methods described here should significantly enhance reverse genetic studies of E. faecium and other closely related species for functional and mechanistic studies. To make it easier to study perturbations of essential genes in E. faecium, I describe my efforts to generate genetic infrastructure to enable the application of CRISPR interference programmed gene silencing in Chapter 4. Here, I demonstrate the generation of a compatible integration vector de novo for large gene insertions for E. faecium. Leveraging my integration system, I create a stably expressing and genome integrated gfp gene in E. faecium Com15 strain. I then demonstrate efficient targeting by Streptococcus pyogenes or Streptococcus thermophilis dead Cas9 (dCas9) of gfp produces ample gene silencing in E. faecium Com15. In Chapter 5, I present foundational experiments and the roadmap to generating functional CRISPRi in vancomycin-resistant E. faecium (VREfm). By utilizing my integration system, I was able to create genome integrated inducible dCas9 controlled by IPTG or agmatine inducible systems in VREfm. Furthermore, I generate and characterize a compact, highly transformable single-guide RNA (sgRNA) delivery vector for CRISPRi assays. In developing CRISPRi for VREfm, I discover a putative anti-CRISPR protein that has CRISPR inhibitory effects in E. faecium Com15. Overall, these experiments provide a detailed roadmap for implementing a CRISPRi system into VREfm. Overall, the projects described here should accelerate the genetic methodological landscape for E. faecium