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Article ; Online: Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Stapleton, Emma M / Thurman, Andrew L / Pezzulo, Alejandro A / Comellas, Alejandro P / Thornell, Ian M

American journal of physiology. Cell physiology

2023 Volume 326, Issue 2, Page(s) C540–C550

Abstract: Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in ... ...

Abstract	Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased
MeSH term(s)	Humans ; Amiloride ; Vitamin D ; Calcitriol/pharmacology ; Epithelial Sodium Channels/genetics ; Epithelial Sodium Channels/metabolism ; Lung/metabolism ; Vitamins ; Vitamin D Deficiency ; RNA, Messenger/genetics
Chemical Substances	Amiloride (7DZO8EB0Z3) ; Vitamin D (1406-16-2) ; Calcitriol (FXC9231JVH) ; Epithelial Sodium Channels ; Vitamins ; RNA, Messenger
Language	English
Publishing date	2023-12-25
Publishing country	United States
Document type	Journal Article
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00369.2023
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Article ; Online: Eosinophil expression of Triggering receptor expressed on myeloid cells-1 (TREM-1) restricts type 2 lung inflammation.

Bowen, Jayden L / Keck, Kathy / Baruah, Sankar / Nguyen, Kathy H / Thurman, Andrew L / Pezzulo, Alejandro A / Klesney-Tait, Julia

Journal of leukocyte biology

2024

Abstract: Asthma affects 25 million Americans and recent advances in treatment are effective for only a portion of severe asthma patients. Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an innate receptor that canonically amplifies inflammatory ... ...

Abstract	Asthma affects 25 million Americans and recent advances in treatment are effective for only a portion of severe asthma patients. Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an innate receptor that canonically amplifies inflammatory signaling in neutrophils and monocytes, plays a central role in regulating lung inflammation. It is unknown how TREM-1 contributes to allergic asthma pathology. Utilizing a murine model of asthma, flow cytometry revealed TREM-1+ eosinophils in the lung tissue and airway during allergic airway inflammation. TREM-1 expression was restricted to recruited, inflammatory eosinophils. Expression was induced on bone marrow derived eosinophils by incubation with IL-33, LPS, or GM-CSF. Compared to TREM-1- airway eosinophils, TREM-1+ eosinophils were enriched for pro-inflammatory gene sets including migration, respiratory burst, and cytokine production. Unexpectedly, eosinophil-specific ablation of TREM-1 exacerbated airway IL-5 production, airway MUC5AC production, and lung tissue eosinophil accumulation. Further investigation of transcriptional data revealed apoptosis and superoxide generation related gene sets were enriched in TREM-1+ eosinophils. Consistent with these findings, Annexin V and Caspase 3/7 staining demonstrated higher rates of apoptosis among TREM-1+ eosinophils compared to TREM-1- eosinophils in the inflammatory airway. In vitro, Trem1/3-/- bone marrow derived eosinophils consumed less oxygen than WT in response to PMA, suggesting that TREM-1 promotes superoxide generation in eosinophils. These data reveal protein level expression of TREM-1 by eosinophils, define a population of TREM-1+ inflammatory eosinophils, and demonstrate that eosinophil TREM-1 restricts key features of type 2 lung inflammation.
Language	English
Publishing date	2024-03-28
Publishing country	England
Document type	Journal Article
ZDB-ID	605722-6
ISSN	1938-3673 ; 0741-5400
ISSN (online)	1938-3673
ISSN	0741-5400
DOI	10.1093/jleuko/qiae061
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Article ; Online: Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells.

Ghimire, Shreya / Stewart, Carley G / Thurman, Andrew L / Pezzulo, Alejandro A

Scientific reports

2021 Volume 11, Issue 1, Page(s) 19438

Abstract: RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. ... ...

Abstract	RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.
MeSH term(s)	Cells, Cultured ; DNA, Complementary/chemical synthesis ; Fibroblasts ; Fludrocortisone ; Gene Expression Profiling/methods ; Humans ; Imidazoles ; Sequence Analysis, RNA/methods
Chemical Substances	DNA, Complementary ; Imidazoles ; calmidazolium (4R9H38JAWL) ; Fludrocortisone (U0476M545B)
Language	English
Publishing date	2021-09-30
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-98912-x
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Article ; Online: Differential gene expression analysis for multi-subject single-cell RNA-sequencing studies with aggregateBioVar.

Thurman, Andrew L / Ratcliff, Jason A / Chimenti, Michael S / Pezzulo, Alejandro A

Bioinformatics (Oxford, England)

2021 Volume 37, Issue 19, Page(s) 3243–3251

Abstract: Motivation: Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful ...

Abstract	Motivation: Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful studies. As scRNA-seq costs have decreased, collecting data from more than one biological replicate has become more feasible, but careful modeling of different layers of biological variation remains challenging for many users. Here, we propose a statistical model for scRNA-seq gene counts, describe a simple method for estimating model parameters and show that failing to account for additional biological variation in scRNA-seq studies can inflate false discovery rates (FDRs) of statistical tests. Results: First, in a simulation study, we show that when the gene expression distribution of a population of cells varies between subjects, a naïve approach to differential expression analysis will inflate the FDR. We then compare multiple differential expression testing methods on scRNA-seq datasets from human samples and from animal models. These analyses suggest that a naïve approach to differential expression testing could lead to many false discoveries; in contrast, an approach based on pseudobulk counts has better FDR control. Availability and implementation: A software package, aggregateBioVar, is freely available on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/aggregateBioVar.html) to accommodate compatibility with upstream and downstream methods in scRNA-seq data analysis pipelines. Supplementary information: Raw gene-by-cell count matrices for pig scRNA-seq data are available as GEO accession GSE150211. Supplementary data are available at Bioinformatics online.
Language	English
Publishing date	2021-03-25
Publishing country	England
Document type	Journal Article
ZDB-ID	1422668-6
ISSN	1367-4811 ; 1367-4803
ISSN (online)	1367-4811
ISSN	1367-4803
DOI	10.1093/bioinformatics/btab337
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Article: Long-COVID improves in 50% of patients after a year in a Midwestern cohort.

Stalker, Grant / Tudas, Rosarie / Garg, Alpana / Graham, Lauren / Thurman, Andrew L / Wiblin, R Todd / Hamzeh, Nabeel / Blount, Robert J / Villacreses, Raul / Zabner, Joseph / Comellas, Alejandro / Cho, Josalyn L / Pezzulo, Alejandro

medRxiv : the preprint server for health sciences

2024

Abstract: Background: Many of those infected with COVID-19 experience long-term disability due to persistent symptoms known as Long-COVID, which include ongoing respiratory issues, loss of taste and smell, and impaired daily functioning.: Research question: ... ...

Abstract	Background: Many of those infected with COVID-19 experience long-term disability due to persistent symptoms known as Long-COVID, which include ongoing respiratory issues, loss of taste and smell, and impaired daily functioning. Research question: This study aims to better understand the chronology of long-COVID symptoms. Study design and methods: We prospectively enrolled 403 adults from the University of Iowa long-COVID clinic (June 2020 to February 2022). Participants provided symptom data during acute illness, symptom progression, and other clinical characteristics. Patients in this registry received a survey containing questions including current symptoms and status since long-COVID diagnosis (sliding status scale, PHQ2, GAD2, MMRC). Those >12 months since acute-COVID diagnosis had chart review done to track their symptomology. Results: Of 403 participants contacted, 129 (32%) responded. The mean age (in years) was 50.17 +/-14.28, with 31.8% male and 68.2% female. Severity of acute covid treatment was stratified by treatment in the outpatient (70.5%), inpatient (16.3%), or ICU (13.2%) settings. 51.2% reported subjective improvement (sliding scale scores of 67-100) since long-COVID onset. Ages 18-29 reported significantly higher subjective status scores. Subjective status scores were unaffected by severity. 102 respondents were >12 months from their initial COVID-19 diagnosis and were tracked for longitudinal symptom persistence. All symptoms tracked had variance (mean fraction 0.58, range 0.34-0.75) in the reported symptoms at the time of long-COVID presentation when compared with patient survey report. 48 reported persistent dyspnea, 23 (48%) had resolved it at time of survey. For fatigue, 44 had persistence, 12 (27%) resolved. Interpretation: Overall, 51.2% respondents improved since their long-COVID began. Pulmonary symptoms were more persistent than neuromuscular symptoms (anosmia, dysgeusia, myalgias). Gender, time since acute COVID infection, and its severity didn't affect subjective status or symptoms. This study highlights recall bias that may be prevalent in other long-COVID research reliant on participant memory.
Language	English
Publishing date	2024-05-01
Publishing country	United States
Document type	Preprint
DOI	10.1101/2024.04.30.24306497
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Article: Lentiviral vectors transduce lung stem cells without disrupting plasticity.

Cooney, Ashley L / Thurman, Andrew L / McCray, Paul B / Pezzulo, Alejandro A / Sinn, Patrick L

Molecular therapy. Nucleic acids

2021 Volume 25, Page(s) 293–301

Abstract: Life-long expression of a gene therapy agent likely requires targeting stem cells. Here we ask the question: does viral vector transduction or ectopic expression of a therapeutic transgene preclude airway stem cell function? We used a lentiviral vector ... ...

Abstract	Life-long expression of a gene therapy agent likely requires targeting stem cells. Here we ask the question: does viral vector transduction or ectopic expression of a therapeutic transgene preclude airway stem cell function? We used a lentiviral vector containing a GFP or cystic fibrosis transmembrane conductance regulator (
Language	English
Publishing date	2021-06-24
Publishing country	United States
Document type	Journal Article
ZDB-ID	2662631-7
ISSN	2162-2531
ISSN	2162-2531
DOI	10.1016/j.omtn.2021.06.010
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Article ; Online: FXYD3 facilitates Na

Cano Portillo, Camilo / Villacreses, Raul / Thurman, Andrew L / Pezzulo, Alejandro A / Zabner, Joseph / Thornell, Ian M

American journal of physiology. Cell physiology

2022 Volume 323, Issue 4, Page(s) C1044–C1051

Abstract: Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 ... ...

Abstract	Na/K ATPase activity is essential for ion transport across epithelia. FXYD3, a γ subunit of the Na/K ATPase, is expressed in the airway, but its function remains undetermined. Single-cell RNA sequencing and immunohistochemistry revealed that FXYD3 localizes within the basolateral membrane of all airway epithelial cells. To study FXYD3 function, we reduced
MeSH term(s)	Amiloride ; Humans ; Membrane Proteins/metabolism ; Neoplasm Proteins/metabolism ; Nystatin ; Ouabain ; RNA, Small Interfering/genetics ; Sodium/metabolism ; Sodium-Potassium-Exchanging ATPase/genetics ; Sodium-Potassium-Exchanging ATPase/metabolism
Chemical Substances	FXYD3 protein, human ; Membrane Proteins ; Neoplasm Proteins ; RNA, Small Interfering ; Nystatin (1400-61-9) ; Ouabain (5ACL011P69) ; Amiloride (7DZO8EB0Z3) ; Sodium (9NEZ333N27) ; Sodium-Potassium-Exchanging ATPase (EC 7.2.2.13)
Language	English
Publishing date	2022-08-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00047.2022
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Article: Parallel Evolution of Linezolid Resistant

Pitcher, Nicholas J / Feder, Andries / Bolden, Nicholas / Zirbes, Christian F / Pamatmat, Anthony J / Boyken, Linda / Hill, Jared J / Thurman, Andrew L / Reeb, Valérie C / Porterfield, Harry S / Moustafa, Ahmed M / Planet, Paul J / Fischer, Anthony J

bioRxiv : the preprint server for biology

2023

Abstract: Background: Linezolid is an antibiotic used to treat serious : Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance.: Methods: We identified ... ...

Abstract	Background: Linezolid is an antibiotic used to treat serious Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. Methods: We identified patients with Main results: Between 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant Conclusions: Linezolid 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 point: Linezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.
Language	English
Publishing date	2023-05-02
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.05.02.539145
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Article ; Online: DNA demethylation fine-tunes IL-2 production during thymic regulatory T cell differentiation.

Teghanemt, Athmane / Misel-Wuchter, Kara / Heath, Jace / Thurman, Andrew / Pulipati, Priyanjali / Dixit, Garima / Geesala, Ramasatya / Meyerholz, David K / Maretzky, Thorsten / Pezzulo, Alejandro / Issuree, Priya D

EMBO reports

2023 Volume 24, Issue 5, Page(s) e55543

Abstract: Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3 is critically dependent upon T cell receptor ( ... ...

Abstract	Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3 is critically dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that ten-eleven translocation (Tet) enzymes, which are DNA demethylases, are required early during double-positive (DP) thymic T cell differentiation and prior to the upregulation of FoxP3 in CD4 single-positive (SP) thymocytes, to promote Treg differentiation. We show that Tet3 selectively controls the development of CD25
MeSH term(s)	Humans ; Mice ; Animals ; Interleukin-2 ; DNA Demethylation ; Thymus Gland ; T-Lymphocytes, Regulatory ; Receptors, Antigen, T-Cell/metabolism ; Cell Differentiation ; Forkhead Transcription Factors/metabolism
Chemical Substances	Interleukin-2 ; Receptors, Antigen, T-Cell ; Forkhead Transcription Factors
Language	English
Publishing date	2023-03-07
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2020896-0
ISSN	1469-3178 ; 1469-221X
ISSN (online)	1469-3178
ISSN	1469-221X
DOI	10.15252/embr.202255543
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Zs.A 5433: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Interleukin 13-Induced Inflammation Increases DPP4 Abundance but Does Not Enhance Middle East Respiratory Syndrome Coronavirus Replication in Airway Epithelia.

Li, Kun / Bartlett, Jennifer A / Wohlford-Lenane, Christine L / Xue, Biyun / Thurman, Andrew L / Gallagher, Thomas M / Pezzulo, Alejandro A / McCray, Paul B

The Journal of infectious diseases

2023 Volume 229, Issue 5, Page(s) 1419–1429

Abstract: Background: Chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that ... ...

Abstract	Background: Chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). Methods: We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine interleukin 13 (IL-13), examining how this affected DPP4 protein levels with MERS-CoV entry and replication. Results: IL-13 exposure for 3 days led to greater DPP4 protein abundance, while a 21-day treatment raised DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly affected by IL-13 treatment. Conclusions: Our results suggest that greater DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13-induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.
MeSH term(s)	Dipeptidyl Peptidase 4/metabolism ; Dipeptidyl Peptidase 4/genetics ; Humans ; Middle East Respiratory Syndrome Coronavirus/physiology ; Interleukin-13/metabolism ; Virus Replication ; Inflammation ; Respiratory Mucosa/virology ; Respiratory Mucosa/metabolism ; Coronavirus Infections/immunology ; Coronavirus Infections/virology ; Virus Internalization ; Epithelial Cells/virology ; Cells, Cultured
Language	English
Publishing date	2023-08-29
Publishing country	United States
Document type	Journal Article
ZDB-ID	3019-3
ISSN	1537-6613 ; 0022-1899
ISSN (online)	1537-6613
ISSN	0022-1899
DOI	10.1093/infdis/jiad383
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