Publications by authors named "Maureen A Kane"

119 Publications

Bacteria induce skin regeneration via IL-1β signaling.

Cell Host Microbe 2021 Mar 26. Epub 2021 Mar 26.

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD 21210, USA; Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21210, USA. Electronic address:

Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1β to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.
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http://dx.doi.org/10.1016/j.chom.2021.03.003DOI Listing
March 2021

Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor.

Cell Biosci 2021 Mar 25;11(1):58. Epub 2021 Mar 25.

Center for Precision Disease Modeling, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.

Background: SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology.

Methods: We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction.

Results: We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells.

Conclusions: Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.
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http://dx.doi.org/10.1186/s13578-021-00568-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993076PMC
March 2021

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Mol Pharm 2021 Apr 23;18(4):1544-1557. Epub 2021 Feb 23.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, United States.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00922DOI Listing
April 2021

Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing.

Biochemistry 2021 Mar 22;60(10):780-790. Epub 2021 Feb 22.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, United States.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.
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http://dx.doi.org/10.1021/acs.biochem.0c00940DOI Listing
March 2021

Retinoic acid production, regulation and containment through Zic1, Pitx2c and Cyp26c1 control cranial placode specification.

Development 2021 Feb 16;148(4). Epub 2021 Feb 16.

Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In , Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain.
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http://dx.doi.org/10.1242/dev.193227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903997PMC
February 2021

Intracellular homocysteine metabolites in SLE: plasma S-adenosylhomocysteine correlates with coronary plaque burden.

Lupus Sci Med 2021 01;8(1)

Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Background And Aims: We hypothesised that intracellular homocysteine and homocysteine metabolite levels in patients with SLE are disproportionately elevated compared with the levels seen in healthy subjects and that they are independently associated with coronary plaque in SLE.

Methods: A liquid chromatography-tandem mass spectrometry absolute quantification assay was used for the determination of six analytes in both plasma and peripheral blood mononuclear cells (PBMCs): homocysteine (Hcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), cystathionine (Cysta) and 5-methyltetrahydrofolate (5m-THF). We then compared intracellular (PBMC) and extracellular (plasma) Hcy and Hcy metabolite (SAM, SAH, Met, Cysta and 5m-THF) concentrations in 10 patients with SLE and in 10 age, sex and ethnicity matched controls. Subjects with a history of diabetes mellitus, cardiovascular disease, hypertension, alcohol consumption in excess of 3 units per day, anaemia, renal insufficiency (serum creatinine >1.5 mg/dL) and pregnancy were excluded. All patients with SLE had two coronary CT angiography studies as screening for occult coronary atherosclerotic disease.

Results: Plasma from patients with SLE had higher levels of Hcy (p<0.0001), SAH (p<0.05), SAM (p<0.001) and lower levels of Met (p<0.05) and Cysta (p<0.001) compared with controls. PBMC intracellular concentrations from patients with SLE had higher levels of Cysta (p<0.05), SAH (p<0.05), SAM (p<0.001) and lower levels of 5m-THF (p<0.001). Plasma SAH showed a positive correlation with total coronary plaque, calcified plaque and non-calcified plaque (p<0.05).

Conclusion: Intracellular concentrations of Hcy metabolites were significantly different between patients with SLE and controls, despite similar intracellular Hcy levels. Plasma SAH was positively correlated with total coronary plaque, calcified plaque and non-calcified plaque.
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http://dx.doi.org/10.1136/lupus-2020-000453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825256PMC
January 2021

Mechanistic Analysis of an Extracellular Signal-Regulated Kinase 2-Interacting Compound that Inhibits Mutant BRAF-Expressing Melanoma Cells by Inducing Oxidative Stress.

J Pharmacol Exp Ther 2021 01 27;376(1):84-97. Epub 2020 Oct 27.

Department of Pharmaceutical Sciences, University of Maryland, Baltimore- School of Pharmacy, Baltimore, Maryland

Constitutively active extracellular signal-regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1-mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound () and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with showed rapid changes in immediate early gene levels, including DEF motif-containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the ROS inhibitor -acetyl cysteine reversed -induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism. SIGNIFICANCE STATEMENT: Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the proliferation and survival of many cancer cell types. Given the diversity of cellular functions regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.
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http://dx.doi.org/10.1124/jpet.120.000266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788356PMC
January 2021

Static Growth Promotes PrrF and 2-Alkyl-4(1)-Quinolone Regulation of Type VI Secretion Protein Expression in Pseudomonas aeruginosa.

J Bacteriol 2020 11 19;202(24). Epub 2020 Nov 19.

University of Maryland, Baltimore, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, Maryland, USA

is an opportunistic pathogen that is frequently associated with both acute and chronic infections. possesses a complex regulatory network that modulates nutrient acquisition and virulence, but our knowledge of these networks is largely based on studies with shaking cultures, which are not likely representative of conditions during infection. Here, we provide proteomic, metabolic, and genetic evidence that regulation by iron, a critical metallonutrient, is altered in static cultures. Specifically, we observed a loss of iron-induced expression of proteins for oxidative phosphorylation, tricarboxylic acid (TCA) cycle metabolism under static conditions. Moreover, we identified type VI secretion as a target of iron regulation in cells under static but not shaking conditions, and we present evidence that this regulation occurs via PrrF small regulatory RNA (sRNA)-dependent production of 2-alkyl-4(1)-quinolone metabolites. These results yield new iron regulation paradigms in an important opportunistic pathogen and highlight the need to redefine iron homeostasis in static microbial communities. Host-mediated iron starvation is a broadly conserved signal for microbial pathogens to upregulate expression of virulence traits required for successful infection. Historically, global iron regulatory studies in microorganisms have been conducted in shaking cultures to ensure culture homogeneity, yet these conditions are likely not reflective of growth during infection. is a well-studied opportunistic pathogen and model organism for iron regulatory studies. Iron homeostasis is maintained through the Fur protein and PrrF small regulatory sRNAs, the functions of which are highly conserved in many other bacterial species. In the current study, we examined how static growth affects the known iron and PrrF regulons of , leading to the discovery of novel PrrF-regulated virulence processes. This study demonstrates how the utilization of distinct growth models can enhance our understanding of basic physiological processes that may also affect pathogenesis.
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http://dx.doi.org/10.1128/JB.00416-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685562PMC
November 2020

Proteomic Evaluation of the Natural History of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Non-human Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing Includes Dysregulation of the Retinoid Pathway.

Health Phys 2020 11;119(5):604-620

University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD.

Exposure to ionizing radiation results in injuries of the hematopoietic, gastrointestinal, and respiratory systems, which are the leading causes responsible for morbidity and mortality. Gastrointestinal injury occurs as an acute radiation syndrome. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the proteome of jejunum from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Jejunum was analyzed by liquid chromatography-tandem mass spectrometry, and pathway and gene ontology analysis were performed. A total of 3,245 unique proteins were quantified out of more than 3,700 proteins identified in this study. Also a total of 289 proteins of the quantified proteins showed significant and consistent responses across at least three time points post-irradiation, of which 263 proteins showed strong upregulations while 26 proteins showed downregulations. Bioinformatic analysis suggests significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. Canonical pathways altered by radiation included GP6 signaling pathway, acute phase response signaling, LXR/RXR activation, and intrinsic prothrombin activation pathway. Additionally, we observed dysregulation of proteins of the retinoid pathway and retinoic acid, an active metabolite of vitamin A, as quantified by liquid chromatography-tandem mass spectrometry. Correlation of changes in protein abundance with a well-characterized histological endpoint, corrected crypt number, was used to evaluate biomarker potential. These data further define the natural history of the gastrointestinal acute radiation syndrome in a non-human primate model of partial body irradiation with minimal bone marrow sparing.
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http://dx.doi.org/10.1097/HP.0000000000001351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541663PMC
November 2020

Proteomics of Non-human Primate Plasma after Partial-body Radiation with Minimal Bone Marrow Sparing.

Health Phys 2020 11;119(5):621-632

University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD.

High-dose radiation exposure results in organ-specific sequelae that occurs in a time- and dose-dependent manner. The partial body irradiation with minimal bone marrow sparing model was developed to mimic intentional or accidental radiation exposures in humans where bone marrow sparing is likely and permits the concurrent analysis of coincident short- and long-term damage to organ systems. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the plasma proteome of non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing with 6 MV LINAC-derived photons at 0.80 Gy min over a time period of 3 wk. The plasma proteome was analyzed by liquid chromatography-tandem mass spectrometry. A number of trends were identified in the proteomic data including pronounced protein changes as well as protein changes that were consistently upregulated or downregulated at all time points and dose levels interrogated. Pathway and gene ontology analysis were performed; bioinformatic analysis revealed significant pathway and biological process perturbations post high-dose irradiation and shed light on underlying mechanisms of radiation damage. Additionally, proteins were identified that had the greatest potential to serve as biomarkers for radiation exposure.
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http://dx.doi.org/10.1097/HP.0000000000001350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541796PMC
November 2020

Evaluation of Plasma Biomarker Utility for the Gastrointestinal Acute Radiation Syndrome in Non-human Primates after Partial Body Irradiation with Minimal Bone Marrow Sparing through Correlation with Tissue and Histological Analyses.

Health Phys 2020 11;119(5):594-603

University of Maryland, School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD.

Exposure to total- and partial-body irradiation following a nuclear or radiological incident result in the potentially lethal acute radiation syndromes of the gastrointestinal and hematopoietic systems in a dose- and time-dependent manner. Radiation-induced damage to the gastrointestinal tract is observed within days to weeks post-irradiation. Our objective in this study was to evaluate plasma biomarker utility for the gastrointestinal acute radiation syndrome in non-human primates after partial body irradiation with minimal bone marrow sparing through correlation with tissue and histological analyses. Plasma and jejunum samples from non-human primates exposed to partial body irradiation of 12 Gy with bone marrow sparing of 2.5% were evaluated at various time points from day 0 to day 21 as part of a natural history study. Additionally, longitudinal plasma samples from non-human primates exposed to 10 Gy partial body irradiation with 2.5% bone marrow sparing were evaluated at timepoints out to 180 d post-irradiation. Plasma and jejunum metabolites were quantified via liquid chromatography-tandem mass spectrometry and histological analysis consisted of corrected crypt number, an established metric to assess radiation-induced gastrointestinal damage. A positive correlation of metabolite levels in jejunum and plasma was observed for citrulline, serotonin, acylcarnitine, and multiple species of phosphatidylcholines. Citrulline levels also correlated with injury and regeneration of crypts in the small intestine. These results expand the characterization of the natural history of gastrointestinal acute radiation syndrome in non-human primates exposed to partial body irradiation with minimal bone marrow sparing and also provide additional data toward the correlation of citrulline with histological endpoints.
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http://dx.doi.org/10.1097/HP.0000000000001348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546578PMC
November 2020

Lack of Cellular Inflammation in a Non-human Primate Model of Radiation Nephropathy.

Health Phys 2020 11;119(5):588-593

Radiation Oncology, University of Maryland, School of Medicine, Baltimore, MD.

Inflammation is commonly cited as a mechanism of delayed effects of acute radiation exposure (DEARE). Confirmation of its presence could provide significant insight to targeted use of treatments or mitigators of DEARE. We sought to quantify the presence of cellular inflammation in kidneys of non-human primates that developed acute and chronic kidney injury after a partial body irradiation exposure. We show herein that cellular inflammation is not found as a component of either acute or chronic kidney injury. Other mechanistic pathways of injury must be sought.
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http://dx.doi.org/10.1097/HP.0000000000001329DOI Listing
November 2020

Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells.

Cells 2020 09 11;9(9). Epub 2020 Sep 11.

Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA.

The accurate estimation and eradication of Human Immunodeficiency Virus (HIV) viral reservoirs is limited by the incomplete reactivation of cells harboring the latent replication-competent virus. We investigated whether the in vitro and in vivo addition of retinoic acid (RA) enhances virus replication and improves the detection of latent virus. Peripheral blood mononuclear cells (PBMCs) from naive and anti-retroviral therapy (ART)-treated SIV-infected rhesus macaques (RMs) were cultured in vitro with anti-CD3/CD28 + IL-2 in the presence/absence of RA. Viral RNA and p27 levels were quantified using RT-qPCR and ELISA, respectively. Viral reservoirs were estimated using the Tat/Rev-Induced Limited Dilution Assay (TILDA) and Quantitative Viral Outgrowth Assay (QVOA). In vitro and in vivo measures revealed that there was also an increase in viral replication in RA-treated versus without RA conditions. In parallel, the addition of RA to either CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin during QVOA and TILDA, respectively, was shown to augment reactivation of the replication-competent viral reservoir in anti-retroviral therapy (ART)-suppressed RMs as shown by a greater than 2.3-fold increase for QVOA and 1 to 2-fold increments for multi-spliced RNA per million CD4 T cells. The use of RA can be a useful approach to enhance the efficiency of current protocols used for in vitro and potentially in vivo estimates of CD4 T cell latent reservoirs. In addition, flow cytometry analysis revealed that RA improved estimates of various viral reservoir assays by eliciting broad CD4 T-cell activation as demonstrated by elevated CD25 and CD38 but reduced CD69 and PD-1 expressing cells.
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http://dx.doi.org/10.3390/cells9092076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565696PMC
September 2020

Quantification of common and planar bile acids in tissues and cultured cells.

J Lipid Res 2020 11 22;61(11):1524-1535. Epub 2020 Jul 22.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Baltimore, MD, USA

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, , in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.
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http://dx.doi.org/10.1194/jlr.D120000726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604731PMC
November 2020

Cigalike electronic nicotine delivery systems e-liquids contain variable levels of metals.

Sci Rep 2020 07 17;10(1):11907. Epub 2020 Jul 17.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA.

Electronic nicotine delivery systems (ENDS) are prefilled, battery-operated products intended to deliver nicotine to the user via an inhaled complex aerosol formed by heating a liquid composed of propylene glycol and glycerol, also referred to as vegetable glycerin and collectively called e-liquid, that contains nicotine and various flavor ingredients. Since their introduction in 2006, the number of ENDS on the market has increased exponentially. Despite their growing ubiquity, the possible health risks associated with ENDS use remain poorly understood. One potential concern is the presence of toxic metals in the e-liquid and aerosol. Herein, we report the evaluation of the metal content in the e-liquids from a series of commercially available cigalike ENDS brands (various flavors) determined using inductively coupled plasma mass spectrometry (ICP-MS) following e-liquid extraction. Each brand of cigalike ENDS was purchased at least three times at retail outlets in the Baltimore, Maryland metropolitan region over a period of six months (September 2017 to February 2018). This allowed for comparison of batch-to-batch variability. Several potentially toxic metals, including lead, chromium, copper, and nickel were detected in the e-liquids. In addition, high variability in metal concentrations within and between brands and flavors was observed . The internal assembled parts of each cartridge were analyzed by X-ray imaging, before dissembling so that the materials used to manufacture each cartridge could be evaluated to determine the metals they contained. Following washing to remove traces of e-liquid, lead, chromium, copper and nickel were all detected in the cigalike ENDS prefilled cartridges, suggesting one potential source for the metals found in the e-liquids. Collectively, these findings can inform further evaluation of product design and manufacturing processes, including quantification of metal concentrations in e-liquids over foreseeable storage times, safeguards against high concentrations of metals in the e-liquid before and after aerosolization (by contact with a metal heating coil), and control over batch-to-batch variability.
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http://dx.doi.org/10.1038/s41598-020-67789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368082PMC
July 2020

MALDI-MSI spatially maps N-glycan alterations to histologically distinct pulmonary pathologies following irradiation.

Sci Rep 2020 07 14;10(1):11559. Epub 2020 Jul 14.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.

Radiation-induced lung injury is a highly complex combination of pathological alterations that develop over time and severity of disease development is dose-dependent. Following exposures to lethal doses of irradiation, morbidity and mortality can occur due to a combination of edema, pneumonitis and fibrosis. Protein glycosylation has essential roles in a plethora of biological and immunological processes. Alterations in glycosylation profiles have been detected in diseases ranging from infection, inflammation and cancer. We utilized mass spectrometry imaging to spatially map N-glycans to distinct pathological alterations during the clinically latent period and at 180 days post-exposure to irradiation. Results identified alterations in a number of high mannose, hybrid and complex N-glycans that were localized to regions of mucus and alveolar-bronchiolar hyperplasia, proliferations of type 2 epithelial cells, accumulations of macrophages, edema and fibrosis. The glycosylation profiles indicate most alterations occur prior to the onset of clinical symptoms as a result of pathological manifestations. Alterations in five N-glycans were identified as a function of time post-exposure. Understanding the functional roles N-glycans play in the development of these pathologies, particularly in the accumulation of macrophages and their phenotype, may lead to new therapeutic avenues for the treatment of radiation-induced lung injury.
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http://dx.doi.org/10.1038/s41598-020-68508-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360629PMC
July 2020

Comparative proteomic analysis of SLC13A5 knockdown reveals elevated ketogenesis and enhanced cellular toxic response to chemotherapeutic agents in HepG2 cells.

Toxicol Appl Pharmacol 2020 09 4;402:115117. Epub 2020 Jul 4.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, United States of America. Electronic address:

Solute carrier family 13 member 5 (SLC13A5) is an uptake transporter mainly expressed in the liver and transports citrate from blood circulation into hepatocytes. Accumulating evidence suggests that SLC13A5 is involved in hepatic lipogenesis, cell proliferation, epilepsy, and bone development in mammals. However, the molecular mechanisms behind SLC13A5-mediated physiological/pathophysiological changes are largely unknown. In this regard, we conducted a differential proteome analysis in HepG2 and SLC13A5-knockdown (KD) HepG2 cells. A total of 3826 proteins were quantified and 330 proteins showed significant alterations (fold change ≥1.5; p < .05) in the knockdown cells. Gene ontology enrichment analysis reveals that 38 biological processes were significantly changed, with ketone body biosynthetic process showing the most significant upregulation following SLC13A5-KD. Catalytic activity and binding activity were the top two molecular functions associated with differentially expressed proteins, while HMG-CoA lyase activity showed the highest fold enrichment. Further ingenuity pathway analysis predicted 40 canonical pathways and 28 upstream regulators (p < .01), of which most were associated with metabolism, cell proliferation, and stress response. In line with these findings, functional validation demonstrated increased levels of two key ketone bodies, acetoacetate and β-hydroxybutyrate, in the SLC13A5-KD cells. Additional experiments showed that SLC13A5-KD sensitizes HepG2 cells to cellular stress caused by a number of chemotherapeutic agents. Together, our findings demonstrate that knockdown of SLC13A5 promotes hepatic ketogenesis and enhances cellular stress response in HepG2 cells, suggesting a potential role of this transporter in metabolic disorders and liver cancer.
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http://dx.doi.org/10.1016/j.taap.2020.115117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398853PMC
September 2020

High concentrations of urinary ethanol metabolites in neonatal intensive care unit infants.

Pediatr Res 2020 12 20;88(6):865-870. Epub 2020 Jun 20.

Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH, USA.

Background: Infants in the neonatal intensive care unit may be exposed to ethanol via medications that contain ethanol as an excipient and through inhalation of ethanol vapor from hand sanitizers. We hypothesized that both pathways of exposure would result in elevated urinary biomarkers of ethanol.

Methods: Urine samples were collected from infants in incubators and in open cribs. Two ethanol metabolites, ethyl sulfate (EtS) and ethyl glucuronide (EtG), were quantified in infants' urine.

Results: A subset of infants both in incubators and open cribs had ethanol biomarkers greater than the cutoff concentration that identifies adult alcohol consumption. These concentrations were associated with the infant having received an ethanol-containing medication on the day of urine collection. When infants who received an ethanol-containing medication were excluded from analysis, there was no difference in ethanol biomarker concentrations between the incubator and crib groups.

Conclusions: Some infants who received ethanol-containing medications had concentrations of ethanol biomarkers that are indicative of adult alcohol consumption, suggesting potential exposure via ethanol excipients.

Impact: Infants and newborns in the neonatal intensive care unit are exposed to concerning amounts of ethanol. No one has shown exposure to ethanol in these infants before this study. The impact is that better understanding of the excipients in medications given to patients in the NICU is needed. When physicians order medications in the NICU, the amount of excipient needs to be indicated.
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http://dx.doi.org/10.1038/s41390-020-1020-5DOI Listing
December 2020

Identifying vitamin A signaling by visualizing gene and protein activity, and by quantification of vitamin A metabolites.

Methods Enzymol 2020 21;637:367-418. Epub 2020 Apr 21.

Stowers Institute for Medical Research, Kansas City, MO, United States; University of Kansas Medical Center, Department of Anatomy and Cell Biology, Kansas City, KS, United States. Electronic address:

Vitamin A (retinol) is an essential nutrient for embryonic development and adult homeostasis. Signaling by vitamin A is carried out by its active metabolite, retinoic acid (RA), following a two-step conversion. RA is a small, lipophilic molecule that can diffuse from its site of synthesis to neighboring RA-responsive cells where it binds retinoic acid receptors within RA response elements of target genes. It is critical that both vitamin A and RA are maintained within a tight physiological range to protect against developmental disorders and disease. Therefore, a series of compensatory mechanisms exist to ensure appropriate levels of each. This strict regulation is provided by a number synthesizing and metabolizing enzymes that facilitate the precise spatiotemporal control of vitamin A metabolism, and RA synthesis and signaling. In this chapter we describe protocols that (1) biochemically isolate and quantify vitamin A and its metabolites and (2) visualize the spatiotemporal activity of genes and proteins involved in the signaling pathway.
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http://dx.doi.org/10.1016/bs.mie.2020.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565286PMC
April 2020

Aberrant retinoic acid production in the decidua: Implications for pre-eclampsia.

J Obstet Gynaecol Res 2020 Jul 28;46(7):1007-1016. Epub 2020 Apr 28.

Department of Gynecology & Obstetrics, Emory University School of Medicine, Atlanta, Georgia, USA.

Fine-tuning of the endometrium during the evanescent 'window of implantation' relies upon an array of diverse and redundant signaling molecules, particularly the ovarian steroids E2 and P4, but also growth factors, eicosanoids, and vitamins including the vitamin A compounds (retinoids). Pregnancy complications such as preeclampsia (PE) can result from aberrations in the production or function of these molecules that arise during this critical period of decidual development. Such aberrations may be reflected by incomplete decidualization, reduced spiral artery modification, and/or loss of immune tolerance to the developing fetus. Our understanding of the role of the active retinoid metabolite all-trans retinoic acid (RA) in maintaining immune balance in certain tissues, along with data describing its role in decidualization, present a compelling argument that aberrant RA signaling in the decidua can play a significant role in the etiology of PE. Recent findings that decidualization and expression of the anti-angiogenic gene product, 'soluble fms-like tyrosine kinase-1' (sFLT1) are negatively correlated and that sFLT1 expression is directly inhibited by RA, provide additional evidence of the critical role of this retinoid in regulating early vascular development in the decidua. This review provides insight into the production and function of RA in the decidua and how modifications in its metabolism and signaling might lead to certain pregnancy disorders such as PE.
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http://dx.doi.org/10.1111/jog.14262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565253PMC
July 2020

Alternatively Activated Macrophages Are the Primary Retinoic Acid-Producing Cells in Human Decidua.

Reprod Sci 2020 01 1;27(1):334-341. Epub 2020 Jan 1.

Department of Gynecology & Obstetrics, Emory University School of Medicine, Atlanta, GA, USA.

In situ production and metabolism of all-trans retinoic acid (RA) in decidual tissue are critically important for endometrial stromal differentiation, embryo implantation, and healthy placentation. However, the cellular source(s) of RA in this tissue has yet to be determined. To identify the primary RA-producing cells in human term decidua, we isolated cells from decidua basalis of delivered placenta and quantified cellular retinal dehydrogenase (RALDH) activity, a major biosynthetic enzyme whose activity determines the synthesis of RA from retinol, using an Aldefluor assay and flow cytometry. RA production in decidual tissue and sorted cell subpopulations was evaluated by liquid chromatography-tandem mass spectrometry. CD14 cells (macrophages/monocytes) showed > 4-fold higher RALDH activity than stromal cells (CD10), T cells (CD3), or non-T lymphocytes (CD3-negative). CD11c cells that did not co-express CD14 showed about one-third the RALDH activity of their CD14 co-expressing counterparts. The highest RALDH activity was found in "alternatively activated" M2 macrophages delineated by the simultaneous expression of CD14 and CD163. The greater RA synthesizing capacity of M2 versus CD14CD163-ve (M1) cells was confirmed by direct quantitation of RA biosynthesis from retinol. RA levels in whole decidua were correlated with M2 cell density but not with stromal cell (CD10) number, the major cell type comprising the decidua. These results identified M2 monocyte/macrophages as the primary source of RA in human term decidua. This finding may have implications for certain pregnancy complications that are known to be associated with reduced numbers of decidual M2 cells.
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http://dx.doi.org/10.1007/s43032-019-00030-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539807PMC
January 2020

Development and bioanalytical method validation of an LC-MS/MS assay for simultaneous quantitation of 2-alkyl-4(1H)-quinolones for application in bacterial cell culture and lung tissue.

Anal Bioanal Chem 2020 Mar 29;412(7):1521-1534. Epub 2020 Jan 29.

Department of Pharmaceutical Sciences, University of Maryland Baltimore, School of Pharmacy, 20 N Pine Street, Baltimore, MD, 21201, USA.

Pseudomonas aeruginosa is an opportunistic pathogen that produces numerous exoproducts during infection that help it evade the host immune system and procure nutrients from the host environment. Among these products are a family of secreted 2-alkyl-4(1H)-quinolone metabolites (AQs), which exhibit a range of biological activities. Here, we describe the validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for quantifying multiple AQ congeners in complex biological matrices. The assay was validated for selectivity, sensitivity, linearity, accuracy, precision, carryover, dilution integrity, recovery, matrix effects, and various aspects of stability (freeze-thaw, bench-top, long-term storage, and autosampler/post-preparative). Using authentic standards for 6 distinct AQ congeners, we report accurate quantitation within a linear range between 25 and 1000 nmol/L for all of the validated AQ standards. This method was successfully applied to quantify AQ concentrations in P. aeruginosa cell culture and in the lungs of mice infected with P. aeruginosa. Further, we confirmed the presence of unsaturated forms of several AQ congeners in cell culture. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-019-02374-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223165PMC
March 2020

Role of Gold in Inflammation and Tristetraprolin Activity.

Chemistry 2020 Feb 16;26(7):1535-1547. Epub 2020 Jan 16.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn St., Baltimore, MD, 21201, USA.

The zinc finger protein tristetraprolin (TTP) regulates inflammation by downregulating cytokine mRNAs. Misregulation results in arthritis, sepsis and cancer, and there is an interest in modulating TTP activity with exogenous agents. Gold has anti-inflammatory properties and has recently been shown to modulate the signaling pathway that produces TTP, suggesting that TTP may be a target of gold. The reactivity of [Au (terpy)Cl]Cl with TTP was investigated by UV/Vis spectroscopy, spin-filter inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy and native electrospray ionization mass spectrometry. Au was found to replace zinc in the protein active site in the reduced Au form, with the Au ion coordinated to two cysteine residues in a linear geometry. The replacement of Zn with Au results in loss of both secondary structure and RNA binding function. In contrast, when Zn TTP is bound to its RNA target, no replacement of Zn with Au is observed, even in the presence of excess Au terpy. This discovery of differential reactivity of gold with TTP versus TTP/RNA offers a potential strategy for selective targeting with gold complexes to control inflammation.
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http://dx.doi.org/10.1002/chem.201904837DOI Listing
February 2020

Galeterone and The Next Generation Galeterone Analogs, VNPP414 and VNPP433-3β Exert Potent Therapeutic Effects in Castration-/Drug-Resistant Prostate Cancer Preclinical Models In Vitro and In Vivo.

Cancers (Basel) 2019 Oct 24;11(11). Epub 2019 Oct 24.

Department of Pharmacology, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201, USA.

These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved prostate cancer drug, enzalutamide (ENZ) with two lead next generation galeterone analogs (NGGAs), VNPP414 and VNPP433-3β, using prostate cancer (PC) in vitro and in vivo models. Antitumor activities of orally administered agents were also assessed in CWR22Rv1 tumor-bearing mice. We demonstrated that Gal and NGGAs degraded AR/AR-V7 and Mnk1/2; blocked cell cycle progression and proliferation of human PC cells; induced apoptosis; inhibited cell migration, invasion, and putative stem cell markers; and reversed the expression of epithelial-to-mesenchymal transition (EMT). In addition, Gal/NGGAs (alone or in combination) also inhibited the growth of ENZ-, docetaxel-, and mitoxantrone-resistant human PC cell lines. The NGGAs exhibited improved pharmacokinetic profiles over Gal in mice. Importantly, in vivo testing showed that VNPP433-3β (at 7.53-fold lower equimolar dose than Gal) markedly suppressed (84% vs. Gal, 47%; < 0.01) the growth of castration-resistant PC (CRPC) CWR22Rv1 xenograft tumors, with no apparent host toxicity. ENZ was ineffective in this CRPC xenograft model. In summary, our findings show that targeting AR/AR-V7 and Mnk1/2 for degradation represents an effective therapeutic strategy for PC/CRPC treatment and supports further development of VNPP433-3β towards clinical investigation.
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http://dx.doi.org/10.3390/cancers11111637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895912PMC
October 2019

Modulation of retinoid signaling: therapeutic opportunities in organ fibrosis and repair.

Pharmacol Ther 2020 01 16;205:107415. Epub 2019 Oct 16.

Medical Sciences Division, Northern Ontario School of Medicine, Sudbury, ON P3E 2C6, Canada; Departments of Chemistry and Biochemistry, and Biology and Biomolecular Sciences Program, Laurentian University, Sudbury, ON, P3E 2C6, Canada. Electronic address:

The vitamin A metabolite, retinoic acid, is an important signaling molecule during embryonic development serving critical roles in morphogenesis, organ patterning and skeletal and neural development. Retinoic acid is also important in postnatal life in the maintenance of tissue homeostasis, while retinoid-based therapies have long been used in the treatment of a variety of cancers and skin disorders. As the number of people living with chronic disorders continues to increase, there is great interest in extending the use of retinoid therapies in promoting the maintenance and repair of adult tissues. However, there are still many conflicting results as we struggle to understand the role of retinoic acid in the multitude of processes that contribute to tissue injury and repair. This review will assess our current knowledge of the role retinoic acid signaling in the development of fibroblasts, and their transformation to myofibroblasts, and of the potential use of retinoid therapies in the treatment of organ fibrosis.
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http://dx.doi.org/10.1016/j.pharmthera.2019.107415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299003PMC
January 2020

Fast liquid chromatography-tandem mass spectrometry method for simultaneous determination of eight antiepileptic drugs and an active metabolite in human plasma using polarity switching and timed selected reaction monitoring.

J Pharm Biomed Anal 2019 Nov 17;176:112816. Epub 2019 Aug 17.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N. Pine Street, Baltimore, MD, 21201, United States. Electronic address:

A fast liquid chromatography-tandem mass spectrometry method using polarity switching and timed selected reaction monitoring has been developed and validated to quantify eight antiepileptic drugs and an active drug metabolite in human plasma within a single analytical assay. The antiepileptic drugs include levetiracetam, lamotrigine, zonisamide, topiramate, carbamazepine, phenytoin, divalproex sodium, oxcarbazepine, and the oxcarbazepine active metabolite 10,11-dihydro-10-hydroxycarbamazepine. The method was validated over concentration ranges specifically for each compound, with a lower limit of quantification of 5-50 ng/mL. The method is accurate and precise, with intra-day and inter-day accuracy ranging from 93.7 to 104.7% and 94.1 to 107.1%, respectively, and intra-day and inter-day coefficient of variation (CV) ranging of 0.8-4.4% and 1.5-7.1%, respectively. Simple two-step protein precipitation and dilution sample preparation, high sensitivity, and high throughput make this method suitable for multi-drug monitoring for epileptic patients.
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http://dx.doi.org/10.1016/j.jpba.2019.112816DOI Listing
November 2019

cPLA2 activation contributes to lysosomal defects leading to impairment of autophagy after spinal cord injury.

Cell Death Dis 2019 07 11;10(7):531. Epub 2019 Jul 11.

Department of Anesthesiology & Center for Shock, Trauma and Anesthesiology Research (STAR), School of Medicine, University of Maryland, Baltimore, MD, USA.

The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.
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http://dx.doi.org/10.1038/s41419-019-1764-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624263PMC
July 2019

Effects of ATP-competitive and function-selective ERK inhibitors on airway smooth muscle cell proliferation.

FASEB J 2019 10 26;33(10):10833-10843. Epub 2019 Jul 26.

Department of Medicine, Jefferson University, Philadelphia, Pennsylvania, USA.

Increased airway smooth muscle (ASM) cell mass and secretory functions are characteristics of airway inflammatory diseases, such as asthma. To date, there are no effective therapies to combat ASM cell proliferation, which contributes to bronchoconstriction and airway obstruction. Growth factors such as platelet-derived growth factor (PDGF) and the activation of the ERK1/2 are major regulators of ASM cell proliferation and airway remodeling in asthma. However, given the ubiquitous expression and multiple functions of ERK1/2, complete inhibition of ERK1/2 using ATP-competitive inhibitors may lead to unwanted off-target effects. Alternatively, we have identified compounds that are designed to target substrate docking sites and act as function-selective inhibitors of ERK1/2 signaling. Here, we show that both function-selective and ATP-competitive ERK1/2 inhibitors are effective at inhibiting PDGF-mediated proliferation, collagen production, and IL-6 secretion in ASM cells. Proteomic analysis revealed that both types of inhibitors had similar effects on reducing proteins related to TGF-β and IL-6 signaling that are relevant to airway remodeling. However, function-selective ERK1/2 inhibitors caused fewer changes in protein expression compared with ATP-competitive inhibitors. These studies provide a molecular basis for the development of function-selective ERK1/2 inhibitors to mitigate airway remodeling in asthma with defined regulation of ERK1/2 signaling.-Defnet, A. E., Huang, W., Polischak, S., Yadav, S. K., Kane, M. A., Shapiro, P., Deshpande, D. A. Effects of ATP-competitive and function-selective ERK inhibitors on airway smooth muscle cell proliferation.
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http://dx.doi.org/10.1096/fj.201900680RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766654PMC
October 2019

Noncoding dsRNA induces retinoic acid synthesis to stimulate hair follicle regeneration via TLR3.

Nat Commun 2019 06 26;10(1):2811. Epub 2019 Jun 26.

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.
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http://dx.doi.org/10.1038/s41467-019-10811-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594970PMC
June 2019