Papers by Kevin Rosenblatt
American Journal of Obstetrics and Gynecology, 2018
<p>The ASK1 signaling complex was co-IP using ASK1 or 14-3-3ζ antibody. Chemical dissociati... more <p>The ASK1 signaling complex was co-IP using ASK1 or 14-3-3ζ antibody. Chemical dissociation of co-precipitated Trx from the complex was monitored in the presence or absence of various disulfide reducing agents in SDS- conditioned buffer as described in the text. DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; THP, tris(hydroxypropyl)phosphine. The experiment was repeated at least twice and a representative Western blot is shown. A representative full blot image showing higher molecular weight protein complexes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141968#pone.0141968.s001" target="_blank">S1 Fig</a></p
<p><sup><i>a</i></sup>Spots 1–3 were identified based on their diff... more <p><sup><i>a</i></sup>Spots 1–3 were identified based on their differential phosphorylation [KO mice (klkl) vs. wild type control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.g002" target="_blank">Fig 2</a>; spots 4 and 5 were downregulated in klkl vs. wild type; spots 6 and 7 were upregulated in klkl mice.</p><p>*expt., experimentally obtained p<i>I</i>s and M<i>r</i>s are those analyzed from 2D gels; deter., determined pIs and Mrs values are based on primary sequence data and were obtained from literature. DHLA, dihydrolipoamide</p><p>Summary of seven protein spots of interest identified by mass spectrometry on 2D gels of Klotho mutant and wild type mice.</p
<p>(A) Plot of native PAGE Western blot of WT1 and <i>kl/kl</i> mice probed wit... more <p>(A) Plot of native PAGE Western blot of WT1 and <i>kl/kl</i> mice probed with 14-3-3ζ antibody. The 14-3-3ζ antibody recognizes a predominant ~30 <i>k</i>Da protein band representing the expected size of a monomer. *p< 0.05, between WT1 and <i>kl/kl</i>. (B) Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. (C) Native PAGE Western blot of WT2 verses EFmKL48 performed under identical conditions described for (A). *p< 0.05, between WT2 and EFmKL48. (D) Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB signals used in the plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.s006" target="_blank">S6</a> Tables.</p
<p>(A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demons... more <p>(A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the Trx/ASK1 complex protection against oxidative stress occurred (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141968#pone.0141968.g001" target="_blank">Fig 1</a>). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 <i>k</i>Da protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141968#pone.0141968.s004" target="_blank">S3 Table</a>.</p
<p>(A) Plots of phosphorylation levels of ProQ diamond stained 14-3-3 proteins. The Pro-Q d... more <p>(A) Plots of phosphorylation levels of ProQ diamond stained 14-3-3 proteins. The Pro-Q diamond stained gel images in Fig 3B (a-d) are close-up views of areas of spots in WT1, <i>kl/kl</i>, WT2 and EFmKL48 full gels respectively within pI 4.3–4.8. Plots are pooled data from two replicate gels and deviations are shown. Representative ProQ diamond gel images and their corresponding positions in SyproRuby-stained protein gels are shown together to normalize for levels of protein load. Digitized values of stained spots used for the plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.s003" target="_blank">S3 Table</a>.</p
PLOS ONE, 2015
<p>(A) RT-PCR profile of Klotho expression relative to GAPDH control. The approximately 630... more <p>(A) RT-PCR profile of Klotho expression relative to GAPDH control. The approximately 630 base pair Klotho product was amplified from normal mouse brain sections using the following specific primers: TGATCAGCGAGCTGGTGCAC (forward), and CCTGTACCTCCAAGTAATCG (reverse). (B) qRT-PCR profile of Klotho expressiondeviations ± SE are shown. (C-D) Immunohistochemical localization of Klotho expression in the SN regions. Details are described in Materials and Methods. Data used for plots in the PCR analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139914#pone.0139914.s008" target="_blank">S8 Table</a>.</p
Genome informatics. International Conference on Genome Informatics, 2005
Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry data ha... more Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry data has been increasingly analyzed for identifying biomarkers to help early detection of the disease. Ovarian cancer commonly recurs at the rate of 75% within a few months or several years later after standard treatment. Since recurrent ovarian cancer is relatively difficult to be diagnosed and small tumors generally respond better to treatment, new methods for the detection of early relapse in ovarian cancer are urgently needed. Here, we propose a new algorithm SVM-MB/RFE (SVM-Markov Blanket/Recursive Feature Elimination) based on SVM-RFE, which identifies biomarkers for predicting the early recurrence of ovarian cancer. In this approach, we first apply t-test for feature pruning and then binning using 5-fold cross validation. Finally, 58 peaks are obtained from 27,000 of the raw data. Such dramatically reduced features relax the computational burden in the next step of our algorithm. We comp...
Frontiers in Molecular Biosciences, 2019
Identification of somatic molecular alterations in primary and metastatic solid tumor specimens c... more Identification of somatic molecular alterations in primary and metastatic solid tumor specimens can provide critical information regarding tumor biology and its heterogeneity, and enables the detection of molecular markers for clinical personalized treatment assignment. However, the optimal methods and target genes for clinical use are still being in development. Toward this end, we validated a targeted amplification-based NGS panel (Oncomine comprehensive assay v1) on a personal genome machine sequencer for molecular profiling of solid tumors. This panel covers 143 genes, and requires low amounts of DNA (20 ng) and RNA (10 ng). We used 27 FFPE tissue specimens, 10 cell lines, and 24 commercial reference materials to evaluate the performance characteristics of this assay. We also evaluated the performance of the assay on 26 OCT-embedded fresh frozen specimens (OEFF). The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. Our results indicate that this is a reliable method to determine molecular alterations in both fixed and fresh frozen solid tumor samples, including core needle biopsies.
Clinical Research (Excluding Clinical Trials), 2019
Introduction: Exosomes have been used as a source of biomarkers in diagnostics for many diseases,... more Introduction: Exosomes have been used as a source of biomarkers in diagnostics for many diseases, including cancer, and are now being used as therapeutic delivery systems. To date, the most efficient method for isolating exosomes from serum or plasma samples has been to use a size exclusion, chromatography (SEC) based isolation. The conditions of the SEC separation can be modified to yield extracellular vesicles of different sizes and diagnostic value. Apoptotic bodies, 50-5000nm, microvesicles, 50-1000nm, and exosomes, 30-120nm, all have different diagnostic and targeted applications. Here, we show a routine and robust method for the isolation and monitoring of proteins in exosomes isolated from plasma samples. The methods and conditions used here are focused on rapidly providing a high yield of exosome vesicles in a monodisperse 40nm size range from a plasma sample. Materials and methods: K2EDTA plasma samples from donor disease and control groups were collected with IRB approval. All SEC columns and reagents for exosome isolation were obtained as a kit (NX Prenatal, Houston, TX). The 40nm exosome fraction from the column was lysed, the proteins reduced, alkylated and digested with trypsin; the subsequent peptides were analyzed by ultra-high performance liquid chromatography (Vanquish Horizon, ThermoFisher Scientific), tandem mass spectrometry (Q-Exactive, ThermoFisher Scientific), to quantify and identify greater than 500 proteins from exosomes, isolated from a single plasma sample, in a 1 hour method. Subsequent isolations of 40nm exosome fractions were labeled with a sulfo-NHS-biotin linker, followed by lysis and enrichment with streptavidin columns, and an identical LC-MS analysis to obtain quantitative analysis of the surface membrane protein contents on the exosomes. Results: Raw data was analyzed using the label-free quantitation module in Proteome Discoverer (Thermofisher Scientific). Peptide peak areas from three technical replicate runs were used to ascertain the reproducibility of the method across both normal and disease samples. Membrane protein extracts LC-MS data was further grouped by Protein Center (ThermoFisher Scientific) by cell type and tissue of origin. Conclusions: The methods shown here provide a robust and efficient platform for 40nm exosome isolation from human plasma samples, and quantitative analysis of the protein content thereof for the purposes of biomarker discovery, quantification and screening purposes. Citation Format: David A. Sarracino, Xiaolei Xie, Courtney E. Martel, Shen Luan, Kevin Rosenblatt. Exosomes in plasma for protein based cancer biomarkers: A clinical research tool using UHPLC-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2225.
Oncotarget, 2018
Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent repo... more Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1...
Addictive Behaviors Reports, 2017
Introduction: Opioid use disorder (OUD) is characterized by a problematic pattern of opioid use l... more Introduction: Opioid use disorder (OUD) is characterized by a problematic pattern of opioid use leading to clinically-significant impairment or distress. Opioid agonist treatment is an integral component of OUD management, and buprenorphine is often utilized in OUD management due to strong clinical evidence for efficacy. However, interindividual genetic differences in buprenorphine metabolism may result in variable treatment response, leaving some patients undertreated and at increased risk for relapse. Clinical pharmacogenomics studies the effect that inherited genetic variations have on drug response. Our objective is to demonstrate the impact of pharmacogenetic testing on OUD management outcomes. Methods: We analyzed a patient who reported discomfort at daily buprenorphine dose of 24 mg, which was a mandated daily maximum by the pharmacy benefits manager. Regular urine screenings were conducted to detect the presence of unauthorized substances, and pharmacogenetic testing was used to determine the appropriate dose of buprenorphine for OUD management. Results: At the 24 mg buprenorphine daily dose, the patient had multiple relapses with unauthorized substances. Pharmacogenetic testing revealed that the patient exhibited a cytochrome P450 3A4 ultrarapid metabolizer phenotype, which necessitated a higher than recommended daily dose of buprenorphine (32 mg) for adequate OUD management. The patient exhibited a reduction in the number of relapses on the pharmacogenetic-based dose recommendation compared to standard dosing. Conclusion: Pharmacogenomic testing as clinical decision support helped to individualize OUD management. Collaboration by key stakeholders is essential to establishing pharmacogenetic testing as standard of care in OUD management.
BioTechniques, 2016
High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering a... more High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of hum...
JCI Insight, 2016
Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessel... more Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.
Molecular Cancer Therapeutics, 2015
Background - A challenge in the analysis of circulating tumor cells (CTCs) is their scarcity and ... more Background - A challenge in the analysis of circulating tumor cells (CTCs) is their scarcity and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs were aggregated into close contact to facilitate interactions and growth in culture. We then demonstrated the ability to perform next generation sequencing (NGS) of the spheroids to detect somatic mutations from renal and prostate cancers. Methods - Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion Biosciences), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then magnetized by incubation with NanoShuttle (NS, Nano3D Biosciences) and print...
Molecular Cancer Therapeutics, 2015
Introduction: Tumor genotyping using fluid samples such as blood can potentially allow tracking o... more Introduction: Tumor genotyping using fluid samples such as blood can potentially allow tracking of dynamic changes in mutational profiles over time and allow better access than biopsies. We present a method to detect somatic mutations from a blood draw, where circulating tumor cell (CTC) enrichment above 10% of total cell numbers allows the use of standard gene panels typically used to analyze tissue-based biopsies. Methods: Clinical samples were obtained from 9 prostate cancer (PC) patients and 6 renal cell cancer (RCC) patients, followed by CTC enrichment using the IsoFluxTM System. Cells were lysed and DNA amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion Biosciences) and quantified via qPCR. CTCs defined as CK+, CD45- nucleated cells (DAPI+) for cell enumeration. Analytical samples were prepare by spiking tumor derived cell lines into whole blood and parallel analysis. Next-generation sequencing was performed using 3 targeted cancer panels on the Ion torre...
PLOS ONE, 2015
The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling... more The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho&amp;amp;amp;amp;amp;amp;amp;amp;#39;s stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.
Frontiers in Pharmacology, 2016
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Papers by Kevin Rosenblatt