Biology subjects

Thurman, A.

Publications and source records attributed to Thurman, A..

8 recordsLinked to original sources

bioRxiv2025

Acute malaria dysregulates specialized lymph node macrophages to suppress vaccine-elicited protection against Ebola virus

The filovirus, Ebola virus (EBOV), causes outbreaks of Ebola virus disease (EVD) throughout equatorial Africa. ERVEBO(R) (rVSV/EBOV) is a replication-competent, recombinant vesicular stomatitis virus (rVSV)-vectored vaccine licensed to control EVD outbreaks. EVD outbreaks occur in regions endemic for Plasmodium-caused malaria. Plasmodium infections persist due in part to the parasites ability to evade sterilizing immunity which also dampens immune responses to heterologous vaccines. Acute murine Plasmodium infection at the time of rVSV/EBOV vaccination reduced vaccine-mediated protection against mouse-adapted EBOV (ma-EBOV) challenge. Decreased protection was associated with a Plasmodium-induced interferon gamma (IFN-{gamma})-mediated decrease of rVSV/EBOV replication in lymph node (LN) macrophages, resulting in reduced primary anti-EBOV glycoprotein antibody responses. Higher doses of rVSV/EBOV partially overcame the antibody deficits and elicited protective responses. Evidence of the negative impact of Plasmodium on the efficacy of low dose rVSV/EBOV vaccine protocols supports the use of high antigen loads in effective management of EVD outbreaks. IMPORTANCEWe show that blood-stage murine Plasmodium infections negatively impacts the primary antibody response elicited by low dose rVSV/EBOV vaccination and results in reduced protection against a lethal dose of ma-EBOV. This defect occurs within the draining lymph node due to the elevation of IFN-{gamma} elicted in Py-infected mice. The Py-imposed decrease in vaccine-mediated protection can be overcome with higher doses of rVSV/EBOV. While the strong protection conferred by rVSV/EBOV and significant side effects known to be associated with this vaccine have led to the suggestion that the vaccine dosage be reduced19, our studies provide a rationale for maintaining a higher dose.

microbiology↗

bioRxiv2025

HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis

mTORC1 is a central regulator of cell growth that is directly activated by the small GTPase Rheb on the lysosomal membrane in response to growth factors. We recently reported that polyubiquitinated Rheb facilitates its interaction with mTORC1, leading to mTORC1 activation. However, the molecular mechanisms underlying the ubiquitination of Rheb and its interaction with mTORC1 for activation are not fully understood. In this study, we demonstrate that HUWE1, an E3 ubiquitin ligase, preferentially interacts with ubiquitinated Rheb and is essential for the interaction between Rheb and mTOR, as well as for mTORC1 activation. Additionally, HUWE1 is necessary for Rheb to interact with CAD, a crucial enzyme involved in de novo pyrimidine biosynthesis, and for its activation through the mTORC1-S6K1 pathway. Knocking down HUWE1 in cultured cells or in the liver tissues of mice inhibits mTORC1 activity without affecting the phosphorylation of Akt or TSC2, nor does it affect the lysosomal localization of TSC2 or mTORC1. Furthermore, HUWE1 specifically enhances the expression of CAD without influencing other enzymes involved in de novo pyrimidine synthesis and maintains UMP levels in the hepatocytes of liver tissues. These findings indicate that HUWE1 serves as a key organizer of the Rheb ubiquitin complex, amplifying mTORC1 activity and playing a vital role in stimulating de novo pyrimidine synthesis by enhancing CAD expression and activity. HighlightsO_LIHUWE1 and CAD preferentially interact with ubiquitinated Rheb. C_LIO_LIHUWE1 further ubiquitinates Rheb and supports Rheb interaction with mTORC1 and CAD. C_LIO_LIHUWE1 stimulates mTORC1 activity through its E3 ligase activity. C_LIO_LIHUWE1 also supports pyrimidine synthesis by enhancing CAD expression. C_LI

biochemistry↗

bioRxiv2023

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis

BackgroundLinezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). ObjectivesThe goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. MethodsWe identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main ResultsBetween 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. ConclusionsLinezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key PointLinezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

microbiology↗

bioRxiv2022

Sex and genetic specific effects on behavioral, but not metabolic, responses to a high fat diet in heterogeneous stock rats

Obesity is a growing epidemic associated with a range of comorbidities, including anxiety and depression. Genetics and environmental factors such as diet contribute to both adiposity and anxiety/depression. Heterogeneous stock (HS) rats are an outbred colony and useful for genetic mapping of complex traits. We have previously shown that HS male rats exhibit worsened metabolic and behavioral health in response to high fat diet (HFD). This study aims to determine if females have similar response to diet and if response to diet interacts with genetic background. We measured multiple metabolic (body weight, fat pad weight, glucose tolerance, fasting glucose and insulin) and behavioral (elevated plus maze, open field test, and forced swim test) outcomes in a large cohort of male and female rats on either HFD or low fat diet (LFD). We estimated overall heritability as well as heritability of response to diet for each outcome. Both sexes showed worsened metabolic measures when fed HFD compared to LFD. In contrast, only males exhibited altered behavioral responses to HFD relative to LFD, with no effect in females. Most metabolic and behavioral measures showed overall heritability in both sexes. In contrast, although there was some evidence for gene by diet (GxD) interactions for behavioral measures in males, GxD interactions were generally not found for the metabolic measures. These data demonstrate an important role of diet, sex and genetics in metabolic and behavioral phenotypes in HS rats, with a potential role of gene by diet interactions for behavioral outcomes only in males.

genetics↗

bioRxiv2021

Measles virus exits human airway epithelia via infectious center sloughing

Measles virus (MeV) is the most contagious human virus, but we do not fully understand why. Unlike most respiratory viruses, MeV does not infect the airway epithelium immediately. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for [~]3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may slough off while preserving epithelial function. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In sloughed infectious centers, ciliary beating persisted and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, modeling the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center sloughing. 5-ethynyl-2-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the extruded infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeVs strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host. AUTHOR SUMMARYMeasles virus (MeV) is a respiratory pathogen that infects millions worldwide each year. Although sometimes mischaracterized as an innocuous childhood disease, measles remains a leading cause of death for children under five. MeV is the most contagious human virus and requires vaccination rates above 90% to maintain herd immunity. Global decreases in vaccination rates over the past ten years contributed to recent, widespread MeV outbreaks. We uncover here a novel mechanism by which MeV exits the human airways that may explain why it is much more contagious than other viruses. We document that infected cells containing cell-associated virus slough en masse from the airway epithelial sheet. These expelled infectious centers are metabolically active and can transmit infection to primary human monocyte-derived macrophages more efficiently than cell-free virus particles. Thus, cell-associated MeV can transmit host-to-host, a new paradigm for efficient respiratory virus transmission.

microbiology↗

bioRxiv2020

Titration of in-cellula affinities of protein-protein interactions

A genetic assay permits simultaneous quantification of two interacting proteins and their bound fraction at the single-cell level using flow cytometry. In-cellula affinities of protein-protein interactions can be extracted from the acquired data through a titration-like analysis. The applicability of this approach is demonstrated on a diverse set of interactions with proteins from different families and organisms and with in-vitro dissociation constants ranging from picomolar to micromolar.

biochemistry↗

bioRxiv2020

Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract

BackgroundZoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. Materials and methodsWe systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FindingsOur results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. InterpretationThese results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrevious studies of ACE2 mRNA transcript abundance in the human respiratory tract have suggested a possible association between ACE2 expression and age, sex, and the presence of comorbidities. However, these studies have provided conflicting results, as well as a lack of protein validation. Previous ACE2 protein studies have been limited by a paucity of lung tissue samples and reports that have produced contradictory results. Added value of this studyUsing a combination of single-cell RNA sequencing and immunohistochemistry, we describe ACE2 expression in the human respiratory tract. Staining protocols were optimized and validated to show consistent apical localization and avoid non-specific staining. We show ACE2 protein is found in subsets of airway cells and is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development for COVID-19, respectively. We show age, sex, and comorbidities do not increase ACE2 protein expression in the human respiratory tract. Implications of all the available evidenceACE2 protein abundance does not correlate with risk factors for severe clinical outcomes, but in some cases showed an inversed relationship. Features driving COVID-19 susceptibility and severity are complex, our data suggests factors other than ACE2 protein abundance as important determinants of clinical outcomes.

cell biology↗

bioRxiv2019

Single-molecule long-read sequencing reveals the chromatin basis of gene expression

Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remains poorly studied at the single-cell level. Here we present a new experimental approach MeSMLR-seq (methyltransferase treatment followed by single-molecule long-read sequencing) for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules, and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding transcription start site for silently- and actively-transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin statuses spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly-heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming.

bioinformatics↗