Biology subjects

Spencer, S.

Publications and source records attributed to Spencer, S..

12 recordsLinked to original sources

bioRxiv2024

Improved gene targeting in vivo using EoHR, a small molecule inhibitor of 53BP1

Precise genome editing by programmable nucleases such as Cas9 has revolutionised medical research by enabling the creation of gene-edited or knock-in mouse models of disease. However, a major limitation of the approach is the inefficient process of homology driven recombination (HDR) from an exogenous DNA repair template. This is because error-prone, 53BP1-dependent non-homologous end joining (NHEJ) predominates at Cas9-induced double strand breaks (DSBs). Here we report the validation of a cell-permeable and non-toxic inhibitor of 53BP1 called EoHR. In vitro, EoHR prevents 53BP1 binding to dimethylated lysine 20 on histone H4, a marker of DSBs. In cells, EoHR prevents localisation of 53BP1 at nuclease mediated DSBs and promotes HDR at a Cas9-induced break. When tested in vivo during mouse Cas9-mediated gene editing, inclusion of EoHR at the time of microinjection more than doubled the recovery of correctly edited mice and halved the time to project success. Our work shows that inhibition of 53BP by EoHR is a simple and robust way to increase HDR at Cas9 breaks that can also dramatically increase the success rates of animal model generation.

biochemistry↗

bioRxiv2022

Profiling of the human intestinal microbiome and bile acids under physiologic conditions using an ingestible sampling device

The spatiotemporal structure of the human microbiome and metabolome reflects and determines regional intestinal physiology and may have implications for disease. Yet, we know little about the distribution of microbes and their products in the gut because of reliance on stool samples and limited access only to some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed and evaluated a safe, ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. The collection of 240 intestinal samples from 15 healthy individuals using the device revealed significant differences between microbes and metabolites present in the intestines versus stool. Certain microbial taxa were differentially enriched, and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Overall, we identified heterogeneous intestinal profiles of bacterial taxa and metabolites indicating that non-invasive multi-regional sampling of the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.

microbiology↗

bioRxiv2021

Myeloid cell-driven nonregenerative pulmonary scarring is conserved in multiple nonhuman primate species regardless of SARS-CoV-2 infection modality

The novel coronavirus SARS-CoV-2 has caused a worldwide pandemic resulting in widespread efforts in development of animal models that recapitulate human disease for evaluation of medical countermeasures, and to dissect COVID-19 immunopathogenesis. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM) of nonhuman primates. Species-specific cohorts of RM and AGM Rhesus macaques (Macaca mulatta, RMs) and African green monkeys (Chlorocebus aethiops, AGMs) were experimentally infected with homologous SARS-CoV-2 by either direct mucosal instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated equivalent infection initially by either exposure route although the magnitude and duration of viral loading was greater in AGMs than that of the RM. Clinical onset was nearly immediate (+1dpi) in mucosally-exposed cohorts whereas aerosol-infected animals began to show signs +7dpi. Myeloid cell responses indicative of the development of pulmonary scarring and extended lack of regenerative capacity in the pulmonary compartment was a conserved pathologic response in both species by either exposure modality. This pathological commonality may be useful in future anti-fibrosis therapeutic evaluations and expands our understanding of how SARS-CoV-2 infection leads to ARDS and functional lung damage.

microbiology↗

bioRxiv2021

Impact of a 7-day homogeneous diet on interpersonal variation in human gut microbiomes and metabolomes

Metabolism of dietary compounds by the gut microbiota generates a vast array of microbiome-dependent metabolites (MDMs), which are highly variable between individuals. The uremic MDMs (uMDMs) phenylacetylglutamine (PAG), p-cresol sulfate (PCS) and indoxyl sulfate (IS) accumulate during renal failure and are associated with poor outcomes. Targeted dietary interventions may reduce toxic MDMs generation; however, it is unclear if interindividual differences in diet or gut microbiome dominantly contribute to MDM variance. Here we use a 7-day homogeneous average American diet to standardize dietary precursor availability in 21 healthy individuals. Notably, the coefficient of variation in three uMDMs of interest, PAG, PCS, and IS (primary outcome), did not significantly decrease. The majority of circulating MDMs maintained variation despite identical diets. These results highlight the highly personalized profile of MDMs and limited contribution of short-term dietary heterogeneity, suggesting that dietary modification may need to be paired with microbial therapies to control MDM profiles.

microbiology↗

bioRxiv2021

Metformin in nucleus accumbens core reduces cue-induced cocaine seeking in male and female rats

This study investigated the potential therapeutic effects of the FDA-approved drug metformin on cue-induced reinstatement of cocaine seeking. Metformin (dimethyl-biguanide) is a first-line treatment for type II diabetes that, among other mechanisms, is involved in the activation of adenosine monophosphate activated protein kinase (AMPK). Cocaine self-administration and extinction is associated with decreased levels of phosphorylated AMPK within the nucleus accumbens core (NAcore). Previously it was shown that increasing AMPK activity in the NAcore decreased cue-induced reinstatement of cocaine seeking. Decreasing AMPK activity produced the opposite effect. The goal of the present study was to determine if metformin in the NAcore reduces cue-induced cocaine seeking in adult male and female Sprague Dawley rats. Rats were trained to self-administer cocaine followed by extinction prior to cue-induced reinstatement trials. Metformin microinjected in the NAcore attenuated cue-induced reinstatement in male and female rats. Importantly, metformins effects on cocaine seeking were not due to a general depression of spontaneous locomotor activity. In female rats, metformins effects did generalize to a reduction in cue-induced reinstatement of sucrose seeking. These data support a potential role for metformin as a pharmacotherapy for cocaine use disorder, but warrant caution given the potential for metformins effects to generalize to a natural reward in female rats.

neuroscience↗

bioRxiv2021

The pigtail macaque (Macaca nemestrina) model of COVID-19 reproduces diverse clinical outcomes and reveals new and complex signatures of disease

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over four million people worldwide as of July 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.

immunology↗

bioRxiv2021

An Orally Available Cathepsin L Inhibitor Protects Lungs Against SARS-CoV-2-Induced Diffuse Alveolar Damage in African Green Monkeys

The COVID-19 pandemic resulted from global infection by the SARS-CoV-2 coronavirus and rapidly emerged as an urgent health issue requiring effective treatments. To initiate infection, the Spike protein of SARS-CoV-2 requires proteolytic processing mediated by host proteases. Among the host proteases proposed to carry out this activation is the cysteine protease cathepsin L. Inhibiting cathepsin L has been proposed as a therapeutic strategy for treating COVID-19. SLV213 (K777) is an orally administered small molecule protease inhibitor that exhibits in vitro activity against a range of viruses, including SARS-CoV-2. To confirm efficacy in vivo, K777 was evaluated in an African green monkey (AGM) model of COVID-19. A pilot experiment was designed to test K777 in a prophylactic setting, animals were pre-treated with 100mg/kg K777 (N=4) or vehicle (N=2) before inoculation with SARS-CoV-2. Initial data demonstrated that K777 treatment reduced pulmonary pathology compared to vehicle-treated animals. A second study was designed to test activity in a therapeutic setting, with K777 treatment (33 mg/kg or 100 mg/kg) initiated 8 hours after exposure to the virus. In both experiments, animals received K777 daily via oral gavage for 7 days. Vehicle-treated animals exhibited higher lung weights, pleuritis, and diffuse alveolar damage. In contrast, lung pathology was reduced in K777-treated monkeys, and histopathological analyses confirmed the lack of diffuse alveolar damage. Antiviral effects were further demonstrated by quantitative reductions in viral load of samples collected from upper and lower airways. These preclinical data support the potential for early SLV213 treatment in COVID-19 patients to prevent severe lung pathology and disease progression.

immunology↗

bioRxiv2020

Culturing of "Unculturable" Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria from Groundwater

The recovery and cultivation of diverse field-related microorganisms from the terrestrial subsurface environment remains a challenge despite recent advances in modern molecular technology. Here we applied natural organic carbon (C), i.e., sediment-derived natural organic matter (NOM) and bacterial cell lysate, to groundwater microbial communities for a 30-day enrichment incubation, followed by conventional direct-plating for isolation. The groundwater was collected from a background well at the Oak Ridge Reservation Field Research Center, Tennessee. As a comparison, we also included enrichments amended with simple organic C sources, including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex natural organic C sources are more effective in enriching diverse bacterial species from groundwater than simple organic C sources. Microcosms amended with simple organic C (glucose, acetate, benzoate, or oleic acid) show significantly lower biodiversity than unamended control and are dominated by only few phyla such as Proteobacteria and Bacteroidetes. In contrast, microcosms amended with complex natural organic C (sediment NOM or bacterial cell lysate) display significantly higher biodiversity, and enrich distinct species from the phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia, Planctomycetes, and Armatimonadetes). Our subsequent isolation efforts from natural organic C-amended enrichments led to 222 purified bacterial isolates representing 5 phyla, 16 orders, and 54 distinct species including candidate novel, rarely cultivated, and undescribed organisms. ImportanceInnovative strategies for recovering bacterial strains representing the true diversity of microbial communities in the terrestrial subsurface would significantly advance the understanding of ecologically critical taxa residing in these ecosystems. In this study, we demonstrate that complex natural organic C that mimic the naturally available resources for microbes encourages the growth of diverse bacteria much more robustly than traditional simplistic organic C sources. Results from this study will substantially advance and improve the design of strategies to effectively cultivate and isolate diverse and novel subsurface microorganisms in the laboratory. Obtaining axenic cultures of the once-unculturable microorganisms will greatly enhance our understanding of microbial physiology, function, and roles in different biogeochemical niches in terrestrial subsurface ecosystems.

microbiology↗

bioRxiv2019

Mitf-family transcription factor function is required within cranial neural crest cells to promote choroid fissure closure

A critical step in eye development is closure of the choroid fissure (CF), a transient structure in the ventral optic cup through which vasculature enters the eye and ganglion cell axons exit. While many factors have been identified that function during CF closure, the molecular and cellular mechanisms mediating this process remain poorly understood. Failure of CF closure results in colobomas. Recently, MITF was shown to be mutated in a subset of human coloboma patients, but how MITF functions during CF closure is unknown. To address this question, zebrafish with mutations in mitfa and tfec, two members of the Mitf-family of transcription factors, were analyzed and their functions during CF closure determined. mitfa;tfec mutants possess severe colobomas and our data demonstrate that Mitf activity is required within cranial neural crest cells (cNCCs) to facilitate CF closure. In the absence of Mitf function, cNCC migration and localization in the optic cup are perturbed. These data shed light on the cellular mechanisms underlying colobomas in patients with MITF mutations and identify a novel role for Mitf function in cNCCs during CF closure. Summary StatementMitf-family transcription factors act within cranial neural crest cells to promote choroid fissure closure. Without Mitf-family function, cNCC localization and function in the CF is disrupted, thus contributing to colobomas.

developmental biology↗

bioRxiv2019

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

The functional validation of genetic variants of uncertain significance (VUS) found in PID patients by next-generation sequencing has traditionally been carried out in model systems that are susceptible to artefact. We use CRISPR correction of primary human T lymphocytes to demonstrate that a specific variant in an IL-6R deficient patient is causative for their condition. This methodology can be adapted and used for variant assessment of the heterogeneous genetic defects that affect T lymphocytes in PID.

immunology↗

bioRxiv2019

Capturing the Diversity of Subsurface Microbiota - Choice of Carbon Source for Microcosm Enrichment and Isolation of Groundwater Bacteria

Improved and innovative enrichment/isolation techniques that yield to relevant isolates representing the true diversity of environmental microbial communities would significantly advance exploring the physiology of ecologically important taxa in ecosystems. Traditionally, either simple organic carbon (C) or yeast extract is used as C source in culture medium for microbial enrichment/isolation in laboratory. In natural environment, however, microbial population and evolution are greatly influenced by the property and composition of natural organic C. In this study, 8 types of organic C sources were fed to intrinsic groundwater microbes collected at Oak Ridge Reservation Field Research Center (ORR-FRC) background site for a 30-day incubation period to investigate the response of indigenous bacterial communities to different C sources. The tested C sources included simple organic C (glucose, acetate, benzoate, oleic acid, and cellulose) that are either traditionally used as C source in bacterial culture medium or present in natural environments; naturally occurring undefined complex C (bacterial cell lysate and sediment-derived natural organic matter (NOM)); as well as vitamin mixture which is a commonly used ingredient in culture medium. Our results clearly indicate that natural complex C substrates served better in enriching diverse bacteria compared to other C sources. Microcosms amended with small organic C (glucose, acetate, benzoate, or oleic acid) showed significantly lower biodiversity than control groups, dominated by only a few phyla of bacteria such as Proteobacteria and Bacteroidetes which are commonly isolated and already have diverse representative isolates, while those amended with natural complex C (cell lysate or NOM) displayed significantly higher biodiversity than control groups, in which three phyla (Verrucomicrobia, Planctomycetes, and Armatimonadetes) that are poorly represented in published culture collections were abundantly enriched. Further isolation of pure bacterial strains from complex C-amended enrichments led to 51 species representing 4 phyla, 13 orders. Furthermore, 5 isolates with low similarities to published strains were considered to be novel. Results from this study will aid in the design of better cultivation and isolation strategy for maximize the diversity of organisms recovered from subsurface environment.

microbiology↗

bioRxiv2018

RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data from Thermo Orbitrap raw files is demonstrated using the RawTools software. RawTools is a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional preview database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library, and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.

bioinformatics↗