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Wijesinghe D.S.,Virginia Commonwealth University | Allegood J.C.,Virginia Commonwealth University | Gentile L.B.,Virginia Commonwealth University | Chalfant C.E.,Virginia Commonwealth University | And 2 more authors.
Journal of Lipid Research | Year: 2010

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/106 cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d 18:1/16:0 with a minority of d 18:1/24:1 and d 18:1/24:0. The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60° C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d 18:1/18:0 C1P, as well as a significant portion of d 18:1/16:0, d 18:1/24:1, and d 18:1/24:0. In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.


Ward K.E.,University of Notre Dame | Bhardwaj N.,University of Illinois at Chicago | Vora M.,Indiana University | Chalfant C.E.,Virginia Commonwealth University | And 3 more authors.
Journal of Lipid Research | Year: 2013

Group IVA cytosolic phospholipase A 2 (cPLA 2), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affi nity for a cationic patch in the C2 domain-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA 2-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specifi city is mediated by Arg 59 , Arg 61 , and His 62 (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confi rm the origin of C1P specifi city, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the fi rst study demonstrating the molecular origin of C1P recognition.Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc..


Lolans K.,Rush University Medical Center | Calvert K.,Alverno Laboratories | Won S.,Rush University Medical Center | Won S.,Hunter Holmes ire Veterans Administration Medical Center | And 2 more authors.
Journal of Clinical Microbiology | Year: 2010

Klebsiella pneumoniae carbapenemase (KPC) production in Gram-negative bacilli is an increasing problem worldwide. Rectal swab surveillance is recommended as a component of infection prevention programs, yet few screening methods are published. We compared detection of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance specimens by 2 methods: (i) inoculation of swabs in tryptic soy broth containing 2 μg/ml imipenem followed by plating to MacConkey agar (MAC) (method 1) and (ii) streaking swabs on MAC onto which a 10-μg ertapenem disk was then placed (method 2). Simulated rectal swab specimens of challenge isolates from a collection of well-characterized K. pneumoniae and E. coli strains and salvage rectal swab specimens collected from patients at 4 different health care facilities over a 7-month period were tested. The gold-standard comparator was blaKPC PCR testing of isolates. Method 1 detected 4/9 (44%) KPC-positive challenge isolates. By method 2, 9/9 KPC-positive challenge isolates exhibited zones of inhibition of ≤27 mm; all KPC-negative isolates exhibited zones of inhibition greater than 27 mm. The sensitivity and specificity of method 1 for detection of KPC-positive K. pneumoniae and E. coli in 149 rectal swab specimens were 65.6% (95% confidence interval [CI], 46.8% to 80.8%) and 49.6% (95% CI, 40.3% to 58.9%), respectively. With method 2, a zone diameter of ≤27 mm had a sensitivity of 97.0% (95% CI, 82.5% to 99.8%) and specificity of 90.5% (95% CI, 83.3% to 94.9%) for detection of KPC in rectal swab specimens. Direct ertapenem disk testing is simpler, more sensitive, and more specific than selective broth enrichment with imipenem for detection of KPC-producing K. pneumoniae and E. coli in surveillance specimens. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Ingersoll K.S.,University of Virginia | Farrell-Carnahan L.,University of Virginia | Cohen-Filipic J.,Virginia Commonwealth University | Heckman C.J.,Fox Chase Cancer Center | And 3 more authors.
Drug and Alcohol Dependence | Year: 2011

Background: Crack cocaine use undermines adherence to highly active antiretroviral therapy (HAART). This pilot randomized clinical trial tested the feasibility and efficacy of 2 interventions based on the Information-Motivation-Behavioral Skill model to improve HAART adherence and reduce crack cocaine problems. Methods: Participants were 54 adults with crack cocaine use and HIV with <90% HAART adherence. Most participants were African-American (82%) heterosexual (59%), and crack cocaine dependent (92%). Average adherence was 58% in the past 2 weeks. Average viral loads (VL) were detectable (log. VL 2.97). The interventions included 6 sessions of Motivational Interviewing plus feedback and skills building (MI+), or Video information plus debriefing (Video+) over 8 weeks. Primary outcomes were adherence by 14-day timeline follow-back and Addiction Severity Index (ASI) Drug Composite Scores at 3 and 6 months. Repeated measure ANOVA assessed main effects of the interventions and interactions by condition. Results: Significant increases in adherence and reductions in ASI Drug Composite Scores occurred in both conditions by 3 months and were maintained at 6 months, representing medium effect sizes. No between group differences were observed. No VL changes were observed in either group. Treatment credibility, retention, and satisfaction were high and not different by condition. Conclusions: A counseling and a video intervention both improved adherence and drug problems durably among people with crack cocaine use and poor adherence in this pilot study. The interventions should be tested further among drug users with poor adherence. Video interventions may be feasible and scalable for people with HIV and drug use. © 2011 Elsevier Ireland Ltd.


Mietla J.A.,Virginia Commonwealth University | Wijesinghe D.S.,Virginia Commonwealth University | Wijesinghe D.S.,Hunter Holmes ire Veterans Administration Medical Center | Hoeferlin L.A.,Virginia Commonwealth University | And 6 more authors.
Journal of Lipid Research | Year: 2013

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK-/- and CERK+/+ mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK -/- cells as compared to CERK+/+ cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK-/- cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK-/- MEFs. Our studies also demonstrate that the CERK-/- mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK-/- cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.


Goehe R.W.,Virginia Commonwealth University | Shultz J.C.,Virginia Commonwealth University | Murudkar C.,Virginia Commonwealth University | Usanovic S.,Virginia Commonwealth University | And 9 more authors.
Journal of Clinical Investigation | Year: 2010

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser 52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.


Shultz J.C.,Virginia Commonwealth University | Vu N.,Virginia Commonwealth University | Shultz M.D.,Virginia Commonwealth University | Mba M.-U.U.,Virginia Commonwealth University | And 4 more authors.
Molecular Cancer Research | Year: 2012

Two splice variants derived from the Bcl-x gene via alternative 5′ splice site selection (5′SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 50SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 μmol/L) or the pan-protein kinase C (PKC) inhibitor Gö6983 (25 μmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells. ©2012 AACR.


Derber C.J.,Eastern Virginia Medical School | Shankaran S.,Hunter Holmes ire Veterans Administration Medical Center
Current Infectious Disease Reports | Year: 2012

Influenza infections cause significant morbidity and mortality throughout the world, and vaccination rates of health-care workers remain well below target goals. Strategies for increasing vaccination rates include mandatory vaccination of health-care workers, mandatory declination, employee incentives, intensive education, increased access to vaccines, and the use of social media to inform employees of the safety and efficacy of vaccination. While these strategies in combination have been shown to be effective in increasing vaccination rates, personal and religious objections, as well as the potential for infringing on individual autonomy, remain challenges in our efforts to bring healthcare worker vaccination rates up to target goals. © Springer Science+Business Media, LLC 2012.


Boundy S.,Virginia Commonwealth University | Zhao Q.,Virginia Commonwealth University | Fairbanks C.,Virginia Commonwealth University | Folgosa L.,Virginia Commonwealth University | And 3 more authors.
Journal of Clinical Microbiology | Year: 2012

Among 23 patients carrying methicillin-resistant Staphylococcus aureus (MRSA) in their anterior nares, 6 (26%) also carried methicillin-susceptible S. aureus (MSSA) as less prevalent flora. In 4 of the 6 patients, the MSSA was unrelated to prevalent MRSA, as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing. However, in two patients, the strains were identical except for the absence of spontaneous staphylococcal cassette chromosome mec (SCCmec). We consider this evidence of spontaneous SCCmec excision in vivo. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Wijesinghe D.S.,Virginia Commonwealth University | Wijesinghe D.S.,Hunter Holmes ire Veterans Administration Medical Center | Brentnall M.,Hematology and Medical Oncology | Mietla J.A.,Virginia Commonwealth University | And 6 more authors.
Journal of Lipid Research | Year: 2014

In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifi cally, fi broblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK mice) and their wild-type littermates (CERK +/+ ) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fi broblasts from CERK +/+ mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK fi broblasts. This observed difference was refl ected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the infl ammatory stage to the peak of the fi broplasia stage (e.g., proliferation and migration of fi broblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fi broblasts isolated from CERK. As the proper migration of fi broblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing. © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

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