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Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes

Dhasmana, Neha; Ram, Geeta; McAllister, Kathleen N; Chupalova, Yulia; Lopez, Peter; Ross, Hope F; Novick, Richard P
The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3
PMID: 34781740
ISSN: 2150-7511
CID: 5103832

Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS)

Ryan, Kamilah; Rose, Rebecca E; Jones, Drew R; Lopez, Peter A
Flow cytometrists have long observed a spectrum of cell-type-specific changes ranging from minor functional defects to outright cell destruction after purification of cells using conventional droplet cell sorters. We have described this spectrum of cell perturbations as sorter induced cellular stress, or SICS (Lopez and Hulspas, Cytometry, 2020, 97, 105-106). Despite the potential impact of this issue and ubiquitous anecdotes, little has been reported about this phenomenon in the literature, and the underlying mechanism has been elusive. Inspired by others' observations (Llufrio et al., Redox Biology, 2018, 16, 381-387 and Binek et al., Journal of Proteome Research, 2019, 18, 169-181), we set out to examine SICS at the metabolic level and use this information to propose a working model. Using representative suspension (Jurkat) and adherent (NIH/3T3) cell lines we observed broad and consistent metabolic perturbations after sorting using a high-speed droplet cell sorter. Our results suggest that the SICS metabolic phenotype is a common cell-type-independent manifestation and may be the harbinger of a wide-range of functional defects either directly related to metabolism, or cell stress response pathways. We further demonstrate a proof of concept that a modification to the fluidic environment (complete media used as sheath fluid) in a droplet cell sorter can largely rescue the intracellular markers of SICS, and that this rescue is not due to a contribution of metabolites found in media. Future studies will focus on characterizing the potential electro-physical mechanisms inherent to the droplet cell sorting process to determine the major contributors to the SICS mechanism.
PMID: 34031988
ISSN: 1552-4930
CID: 4888802

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A

Radke, Emily E; Li, Zhi; Hernandez, David N; El Bannoudi, Hanane; Kosakovsky Pond, Sergei L; Shopsin, Bo; Lopez, Peter; Fenyö, David; Silverman, Gregg J
Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.
PMID: 33995388
ISSN: 1664-3224
CID: 4876542

Empowering Team Science 101: Collaborating for Reproducible Outcomes Across Diverse Technologies

Lopez, Peter; Schock-Kuster, Elke
Team science is a collaborative effort that leverages the strengths and expertise of professionals trained in diverse fields to solve a scientific research problem. Successful outcomes require an increasingly complex multifactorial translation of research driven by innovations and breakthroughs in technology. With deep expertise in the strengths and limitations of their specific technology and applications, core scientists represent competence-based trust, delivering scientific expertise through unbiased acquisition and transparent processes, ensuring data provenance. Collaborative cores excel at connective thinking to expedite optimization for robust scientific experimental design and results. In this session, we will hear from four experts representing their areas of expertise (Cytometry, Imaging, Genomics, Proteomics & Informatics) who will share the essential information required to facilitate successful collaborations.
PMID: 32831767
ISSN: 1943-4731
CID: 4586812

Evaluating the Effects of Cell Sorting on Gene Expression

Box, Andrew; DeLay, Monica; Tighe, Scott; Chittur, Sridar V; Bergeron, Alan; Cochran, Matthew; Lopez, Peter; Meyer, E Michael; Saluk, Alan; Thornton, Sherry; Brundage, Kathleen
Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.
PMID: 32831654
ISSN: 1943-4731
CID: 4586792

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

Lucas, Fabienne; Gil-Pulido, Jesus; LaMacchia, John; Preffer, Fred; Wallace, Paul K; Lopez, Peter
Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peer-review and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings. © 2019 International Society for Advancement of Cytometry.
PMID: 31769204
ISSN: 1552-4930
CID: 4215852

Imaging in Flow in the Correlation System

Lopez, Peter
The vast majority of flow cytometers in use since the inception of this technology operate by making cellular measurements derived from a pulse of fluorescence or laser-light scatter generated as a particle intercepts a focused laser in a flow system. In the case of fluorescence, these "zero resolution" systems accurately quantify the amount of light without knowledge of the location or distribution of the source of this light within the cell, since the laser focal point typically is larger than the cell being measured. There are situations where the spatial distribution of fluorescence in a cell can provide important additional information, but this information is not available in most contemporary zero resolution flow cytometers. In the early 70s, there was interest in developing rapid automated diagnostic systems for the fields of hematology and cancer diagnostics based on either flow cytometry or microscopic image analysis. In the lab of Leon Wheeless at University of Rochester, scientists and engineers set forth to utilize Stanley Patten's diagnostic criteria in cytopathology towards the development of an automated pre-screening system for detection of atypical or malignant cells in cytologic samples derived from cervical sampling or urine. The principle of concept of slit-scanning, or restricting the detection of light from a cell by sampling through a slit mechanically moved over the image of the cell, was initially tested with a custom fluorescence microscope. This proof of concept led to the development of a slit-scan flow cytometer, where the laser illumination field was narrowed to a plane of laser light, oriented orthogonal to the direction of cell flow, with a thickness much smaller than that of the cell. By evaluating the fluorescence intensity pulse shape in real time, this technique proved highly effective at detecting atypical or malignant cells in a flow cytometer. It was noted with this system, however, that there was an unacceptable false positive rate, although only slightly higher than that obtained using microscopic screening by trained cytotechnologists. In order to evaluate cells in flow and their correlated real-time classification as normal or abnormal, a correlation system was built which provided images in flow of cells correlated with their pulse shape analysis and classification. This presentation will describe the characteristics and operation of this original imaging flow cytometer.
ISSN: 1943-4731
CID: 4354282

Cyt-Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops

Czechowska, Kamila; Lannigan, Joanne; Aghaeepour, Nima; Back, Jessica B; Begum, Julfa; Behbehani, Greg; Bispo, Cláudia; Bitoun, Daniel; Fernández, Alfonso Blanco; Boova, Samuel Tony; Brinkman, Ryan Remy; Ciccolella, Christopher O; Cotleur, Bunny; Davies, Derek; Dela Cruz, Gelo Victoriano; Del Rio-Guerra, Roxana; Des Lauriers-Cox, Ann Marie; Douagi, Iyadh; Dumrese, Claudia; Bonilla Escobar, Diana L; Estevam, Jose; Ewald, Christina; Fossum, Anna; Gaudillière, Brice; Green, Cherie; Groves, Christopher; Hall, Christopher; Haque, Yasmin; Hedrick, Michael Nathan; Hogg, Karen; Hsieh, Elena W Y; Irish, Jonathan; Lederer, James; Leipold, Michael; Lewis-Tuffin, Laura J; Litwin, Virginia; Lopez, Peter; Nasdala, Ines; Nedbal, Jakub; Ohlsson-Wilhelm, Betsy M; Price, Kylie M; Rahman, Adeeb H; Rayanki, Radhika; Rieger, Aja M; Robinson, J Paul; Shapiro, Howard; Sun, Yongliang Steve; Tang, Vera A; Tesfa, Lydia; Telford, William G; Walker, Rachael; Welsh, Joshua A; Wheeler, Paul; Tárnok, Attila
PMID: 31833655
ISSN: 1552-4930
CID: 4601462

Survey on Scientific Shared Resource Rigor and Reproducibility

Knudtson, Kevin L; Carnahan, Robert H; Hegstad-Davies, Rebecca L; Fisher, Nancy C; Hicks, Belynda; Lopez, Peter A; Meyn, Susan M; Mische, Sheenah M; Weis-Garcia, Frances; White, Lisa D; Sol-Church, Katia
Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.
PMID: 31452645
ISSN: 1943-4731
CID: 4054312

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes

Amlani, Bhishma; Liu, Yiyuan; Chen, Taotao; Ee, Ly-Sha; Lopez, Peter; Heguy, Adriana; Apostolou, Effie; Kim, Sang Yong; Stadtfeld, Matthias
The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.
PMID: 29420174
ISSN: 2211-1247
CID: 2947822