Laxminarayana Korutla - Academia.edu (original) (raw)

Papers by Laxminarayana Korutla

Research paper thumbnail of Peripheral Blood Extracellular Vesicle RNA Profiling: The Next Step in Cancer Liquid Biopsies

Annals of surgical oncology, May 16, 2024

Research paper thumbnail of Cell cycle-dependent modulation of alpha-interferon-inducible gene expression and activation of signaling components in Daudi cells

Journal of Biological Chemistry, Oct 1, 1994

Research paper thumbnail of The POZ/BTB protein NAC1 interacts with two different histone deacetylases in neuronal-like cultures

Journal of Neurochemistry, Jun 15, 2005

NAC1 is a cocaine-regulated POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad comple... more NAC1 is a cocaine-regulated POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) protein. NAC1 is increased by cocaine selectively in the nucleus accumbens, a CNS region important for drug addiction. NAC1's role in the cell, however, is not known. Each of the two NAC1 isoforms, sNAC1 (short NAC1) and lNAC1 (long NAC1), may serve as corepressors for other POZ/BTB proteins. This study investigated whether sNAC1 and lNAC1 demonstrated proteinprotein interactions with other corepressors. Histone deacetylase (HDAC) inhibition reversed sNAC1 and lNAC1 repression of Gal4 luciferase, but only in neuronal-like cultures. Because these inhibitors do not distinguish among histone deacetylases, two histone deacetylases were selected for further study. HDAC 3 and 4 both demonstrated protein-protein interactions with sNAC1 and lNAC1. This was shown using coimmunoprecipitations, glutathione-S-transferase (GST) pulldowns and mammalian two-hybrids. Importantly, either the POZ domain or NAC1 without the POZ domain can bind these two HDACs. Other corepressors, specifically NCoR (nuclear receptor corepressor), SMRT (silencing mediator for retinoid and thyroid hormone receptor) and mSin3a, do not exhibit protein-protein interactions with sNAC1 and lNAC1. None showed protein-protein interactions in GST pulldowns or mammalian two-hybrids. Taken together, the results of these experiments indicate sNAC1 and lNAC1 recruit histone deacetylases for transcriptional repression, further enhancing POZ/BTB protein mediated repression.

Research paper thumbnail of Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin

Carcinogenesis, 1995

We explored the regulation of epidermal growth factor (EGF)-mediated activation of EGF receptor (... more We explored the regulation of epidermal growth factor (EGF)-mediated activation of EGF receptor (EGF-R) phosphorylation by curcumin (diferuloyl-methane), a recently identified kinase inhibitor, in cultured NIH 3T3 cells expressing human EGF-R. Treatment of cells with a saturating concentration of EGF for 5-15 min induced increased EGF-R tyrosine phosphorylation by 4- to 11-fold and this was inhibited in a dose- and time-dependent manner by up to 90% by curcumin, which also inhibited the growth of EGF-stimulated cells. There was no effect of curcumin treatment on the amount of surface expression of labeled EGF-R and inhibition of EGF-mediated tyrosine phosphorylation of EGF-R by curcumin was mediated by a reversible mechanism. In addition, curcumin also inhibited EGF-induced, but not bradykinin-induced, calcium release. These findings demonstrate that curcumin is a potent inhibitor of a growth stimulatory pathway, the ligand-induced activation of EGF-R, and may potentially be useful in developing anti-proliferative strategies to control tumor cell growth.

Research paper thumbnail of Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells

PubMed, 1997

Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveo... more Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveolar cells secrete surfactant in response to such secretagogs as terbutaline, calcium (Ca) ionophores (e.g., ionomycin [Io]), and adenosine triphosphate (ATP). A cell membrane receptor for SP-A, one of the surfactant proteins, regulates secretion by negative feedback. We used quantitative fluorescence microscopy to study the effects of SP-A on alterations in cytosolic Ca2+ ([Ca2+]i) elicited by surfactant secretagogs. Freshly isolated type II cells were loaded with Fura-2, then treated with secretagog, in the presence or absence of SP-A. Io and ATP produced biphasic increases in cytosol [Ca2+]i, reflecting first Ca2+ release from intracellular stores, and then influx through the cell membrane. Thapsigargin (TG) and Io directly initiate Ca2+ release; ATP elicits Ca2+ release via receptor-mediated mechanisms. Ca2+ release causes cell membrane Ca channels to open by as yet poorly understood mechanisms. Io itself acts as an additional Ca2+ channel. SP-A blocks much of the Ca2+ release and some of the Ca2+ influx elicited by these secretagogs. Antibody against SP-A receptor restores secretagog-induced Ca2+ fluxes from inhibition by SP-A, confirming that the inhibitory activity of SP-A is mediated through its receptor. Type II cells incubated in Ca2+-free medium plus SP-A show diminished Ca2+ release responses to TG or ATP, suggesting that the action of SP-A to prevent secretagog initiated increases in [Ca2+]i may reflect its ability to block Ca2+ release from cytoplasmic Ca stores. The feedback inhibition of surfactant secretion by SP-A may, correspondingly, be a manifestation of this effect. Because recent work suggests that TGF-beta also inhibits Ca2+ fluxes, SP-A and TGF-beta could be representative of a group of physiologic regulators that act by modulating intracellular Ca signaling.

Research paper thumbnail of Activity-dependent subcellular localization of NAC1

European Journal of Neuroscience, Jul 21, 2005

The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rat... more The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain.

Research paper thumbnail of Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes

Nature metabolism, Sep 7, 2020

Nature MetabolisM Extended Data Fig. 6 | Based on human-into-mouse islet transplant model, immuno... more Nature MetabolisM Extended Data Fig. 6 | Based on human-into-mouse islet transplant model, immunohistochemical profiling of islets ± IVM for markers of angiogenesis (VeGF), anti-apoptosis (Bcl-2, GLP-1) and endothelial cells (VWF) highlights the increased intensity and stained area of these epitopes in the IVM + group. The proportion and intensity of staining, quantified by QuPath using five automated regions of interest (ROIs) per sectioned image, is plotted below each section (* denotes p < 0.05 and ** denotes p < 5x10-5 based on the one-way Kruskal-Wallis H Test; arrow in the GLP-1 IVMsection denotes staining artifact). The particular significant p-values were-VEGF at POD 1 (p=0.009), Bcl-2 at POD1 (p=7x10-8) and POD10 (p=10-9), GLP-1 at POD1 (p=10-8) and POD 10 (p=4x10-9) and VWF at POD 10 (p=2x10-4).

Research paper thumbnail of Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium

Science Translational Medicine, May 22, 2019

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells with... more The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit + cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardio-pulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial †

Research paper thumbnail of Activation of surfactant protein-B transcription: Signaling through the SP-A receptor utilizing the PI3 kinase pathway

Journal of Cellular Physiology, 2000

This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and t... more This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and the means by which it activates transcription of surfactant protein-B. Pulmonary surfactant is a mixture of lipids and associated proteins produced by type II pneumocytes. Interaction of SP-A with its cognate receptor (SPAR) on type II cells is involved in regulating surfactant secretion. This interaction also increases transcription of surfactant proteins and several other genes. To study SP-A cytokine activity, we used as a model surfactant-protein (SP-B) transcription, the activators of which have been characterized. HNF-3 and TTF-1 transcription factors are known to stimulate SP-B transcription. SP-A caused increased phosphorylation and nuclear localization of both. Corresponding increases in protein binding to the SP-B promoter were demonstrated by gel shift analysis. SP-A increased protein binding to HNF-3 and TTF-1 consensus recognition elements. Footprinting analysis indicated that SP-A-induced protein binding to SP-B promoter was greater in amount, but not different in location, from that seen in control cells, which normally transcribe SP-B. SP-A caused transient increases in PI3 kinase localization at the plasma membrane, and SP-A signaling to elicit increased SP-B transcription was blocked by LY294002, an inhibitor of PI3 kinase. Therefore, SP-A signals through PI3 kinase to increase SP-B transcription in type II pneumocytes by enhancing TTF-1 and HNF-3 activation of the SP-B promoter. SP-A activation of this signaling pathway, which affects many cellular functions and has not previously been implicated in type II cell transcriptional activity, has profound import for understanding type II cell biology.

Research paper thumbnail of SP-A as a cytokine: Surfactant protein-A-regulated transcription of surfactant proteins and other genes

Journal of Cellular Physiology, Mar 1, 1999

Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alv... more Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alveolar type II cells that is necessary for normal lung function. Surfactant secretion and reuptake by type II cells are regulated in part by interaction of surfactant protein-A (SP-A) with a specific receptor (SPAR) on type 11 cells. Several chemicals and hormones affect both surfactant secretion and also surfactant gene expression, but consequences of SP-A-SPAR interaction beyond regulating surfactant secretion and reuptake are unknown. Accordingly, we studied the effects of SP-A on surfactant protein gene transcription, mRNA levels, and transcript stability. SP-A elicited new transcription of surfactant proteins SP-A, SP-B, and SP-C and SPAR and c-Jun but had no effect on beta-actin or c-fos transcription. Antibody against SP-A receptor blocked SP-A-induced transcription, confirming that these actions of SP-A were receptor-mediated. SP-A effects on overall transcript levels were more complex. However, SP-A, SP-B, and SP-C mRNA levels doubled in SP-A-treated cells compared to controls. SP-A is known to stabilize surfactant, control its secretion and reuptake by type II cells, and augment host antimicrobial defenses. These data indicate that SP-A also acts as an autocrine cytokine: it binds its receptor and specifically regulates transcription of surfactant proteins and other genes.

Research paper thumbnail of NAC-1 Is a Brain POZ/BTB Protein That Can Prevent Cocaine-Induced Sensitization in the Rat

The Journal of Neuroscience, Aug 15, 2000

Levels of the mRNA NAC-1 are increased in the rat forebrain weeks after cocaine exposure. This lo... more Levels of the mRNA NAC-1 are increased in the rat forebrain weeks after cocaine exposure. This long-term neuroadaptation occurs during the expression of behavioral sensitization, a model of psychostimulant-induced paranoia. NAC-1, the protein encoded by this cocaine-regulated mRNA, contains a Pox virus and zinc finger/bric-a-brac tramtrack broad complex (POZ/BTB) motif, which mediates interactions among several transcriptional regulators. The present studies demonstrate that NAC-1 acts as a transcription factor. NAC-1 was localized to the nucleus of neurons in the brain. Transfection of NAC-1 in cell culture repressed transcription of a reporter gene. NAC-1 was also able to affect the actions of other POZ/BTB proteins in mammalian two-hybrid studies; these interactions required the presence of the POZ/BTB domain. However, NAC-1 appears to be a unique POZ/BTB transcriptional regulator because it does not contain any zinc finger regions found in these other DNA-binding proteins. Adenoviral-mediated overexpression of NAC-1 protein in the rat nucleus accumbens prevented the development but not the expression of behavioral sensitization produced by repeated administration of cocaine. Thus, NAC-1 may modify the longterm behaviors of psychostimulant abuse by regulating gene transcription in the mammalian brain.

Research paper thumbnail of 905: Release of placental exosomes is reduced in spontaneous preterm births

American Journal of Obstetrics and Gynecology, 2020

cannabinoid receptor interacting protein (CRIP) in laboring and non-laboring patients. STUDY DESI... more cannabinoid receptor interacting protein (CRIP) in laboring and non-laboring patients. STUDY DESIGN: Myometrial and placental tissue samples were collected in laboring and non-laboring women undergoing cesarean delivery. Tissue samples were rinsed and frozen at-80 C immediately after delivery. CB1 receptor and CRIP presence and quantification were evaluated using western blot and reverse transcription quantitative polymerase chain reaction. RESULTS: 20 low-risk patients were enrolled. The median gestational age was 38 weeks, 5 days in the labor group (n¼9) and 39 weeks in the non-labor group (n¼11). CB1 receptor presence was identified in myometrial and placental samples. Myometrial and placental tissue quantification showed no significant difference in CB1 receptor expression for all labor compared to non-labor patients (p¼0.4063) or in term spontaneous labor compared to non-labor patients (p¼0.7967). CRIP was identified with no difference in gene expression found in all myometrial (p¼0.2179) or placental (p¼0.7620) labor compared to non-labor tissues. Evaluation of term spontaneous labor compared to term non-labor patients showed significantly less CRIP gene expression in the myometrium of laboring patients (p¼0.0060). CONCLUSION: CB1 receptor and CRIP are present in human myometrial and placental tissues and may play a role in parturition. This role appears to be mediated by the cannabinoid receptor interacting protein.

Research paper thumbnail of Mechanism of interferon action: in vivo activation of 91 kDa transcription factor

PubMed, Sep 1, 1996

Interferons are a family of secreted polypeptides with distinct biological effects. These effects... more Interferons are a family of secreted polypeptides with distinct biological effects. These effects include the regulation of expression of specific cellular genes, antiviral properties, and inhibition of cell growth and proliferation. We investigated the effect of sodium orthovanadate (vanadate), an inhibitor of protein-phosphotyrosine phosphatases, on early biochemical events associated with the stimulation of transcription factor p91 activation by interferon-alpha (IFN alpha) in live human cells. We report that the treatment of cells with vanadate selectively potentiated (2-3 fold) the levels of IFN-stimulated tyrosine phosphorylation of p91 (and not of p113) as compared to the levels of p91 activated by IFN alone and that this was associated with the increased accumulation of phosphorylated p91 in the nucleus, and the activation of protein tyrosine kinases that phosphorylate p91. These results suggest the possible involvement of a vanadate sensitive protein-tyrosine phosphatase(s) in the deactivation of phosphorylated p91 in live human cells.

Research paper thumbnail of Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

Thrombosis and Haemostasis, 1994

Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in ra... more Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodinated and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivatized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.

Research paper thumbnail of Unlocking the Potential of Induced Pluripotent Stem Cells for Wound Healing: The Next Frontier of Regenerative Medicine

Advances in wound care, Nov 1, 2022

SIGNIFICANCE Non-healing wounds are a significant burden for the healthcare system all over the w... more SIGNIFICANCE Non-healing wounds are a significant burden for the healthcare system all over the world. Existing treatment options are not enough to promote healing, highlighting the urgent need for improved therapies. In addition, the current advancements in tissue-engineered skin constructs and stem cell-based therapies are facing significant hurdles due to the absence of a renewable source of functional cells. Recent Advances: Induced pluripotent stem cell technology (iPSC) is emerging as a novel tool to develop the next generation of personalized medicine for the treatment of chronic wounds. The iPSC provides unlimited access to various skin cells to generate complex personalized three-dimensional (3D) skin constructs for disease modeling and autologous grafts. Furthermore, the iPSC-based therapies can target distinct wound healing phases and have shown accelerating wound closure by enhancing angiogenesis, cell migration, tissue regeneration, and modulating inflammation. CRITICAL ISSUES Since the last decade iPSC has been revolutionizing the field of wound healing and skin tissue engineering. Despite the current progress, safety and heterogeneity among iPSC lines are still major hurdles in addition to the lack of large animal studies. These challenges need to be addressed before translating an iPSC-based therapy to the clinic. FUTURE DIRECTIONS Future considerations should be given to performing large animal studies to check the safety and efficiency of iPSC-based therapy in a wound healing setup. Furthermore, strategies should be developed to overcome variation between hiPSC lines, develop an efficient manufacturing process for iPSC-derived products, and generate complex skin constructs with vasculature and skin appendages.

Research paper thumbnail of Activity-dependent subcellular localization of NAC1

European Journal of Neuroscience, 2005

The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rat... more The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain.

Research paper thumbnail of Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection

American Journal of Transplantation

Research paper thumbnail of Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

Thrombosis and Haemostasis, 1994

SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activit... more SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodina- ted and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivitized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening tha...

Research paper thumbnail of Donor Lung Specific Exosome Profiles for Noninvasive Monitoring of Acute Rejection in a Rat Orthotopic Left Lung Transplant Model

The Journal of Heart and Lung Transplantation, 2018

Purpose: There is a critical need for development of biomarkers to monitor for lung transplant re... more Purpose: There is a critical need for development of biomarkers to monitor for lung transplant rejection. We investigated the biomarker potential of circulating donor lung specific exosome profiles for time sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods: Left lung from Wistar transgenic rat expressing exosome marker human specific CD63-GFP was transplanted into Lewis recipient across full major histocompatibility complex mismatch (n= 12). Recipient blood was collected daily (days 0 to 8) and plasma was processed for exosome isolation by size exclusion chromatography and ultracentrifugation. Donor lung specific exosomes were profiled using anti-human CD63 antibody quantum dot on the nanoparticle detector. Fluorescence microscopy for GFP was performed to understand donor lung exosome trafficking during the acute rejection process.

Research paper thumbnail of Intramyocardial cell-based therapy during bidirectional cavopulmonary anastomosis for hypoplastic left heart syndrome: The ELPIS phase I trial

ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstructi... more ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction for the right ventricle (RV) to provide systemic circulation. This leads to substantially increased loads on the RV, wall stress, maladaptive remodeling and dysfunction, which in turn can increase risk of death or transplantation.ObjectivesWe conducted a phase I multicenter trial to assess safety and feasibility of intra-operative MSC injection in HLHS patients to boost RV performance in the systemic position.MethodsAllogeneic MSCs were directly administered by intramyocardial injections during the second stage palliative operation. The primary endpoint was safety.ResultsTen patients received intramyocardial injections of allogeneic MSCs (Lomecel-B). No patients experienced major adverse cardiac events (MACE). All subjects were alive and transplant-free at 1 year following, and experienced growth comparable to healthy control historical data. Cardiac magnetic resonance imaging (CMR) r...

Research paper thumbnail of Peripheral Blood Extracellular Vesicle RNA Profiling: The Next Step in Cancer Liquid Biopsies

Annals of surgical oncology, May 16, 2024

Research paper thumbnail of Cell cycle-dependent modulation of alpha-interferon-inducible gene expression and activation of signaling components in Daudi cells

Journal of Biological Chemistry, Oct 1, 1994

Research paper thumbnail of The POZ/BTB protein NAC1 interacts with two different histone deacetylases in neuronal-like cultures

Journal of Neurochemistry, Jun 15, 2005

NAC1 is a cocaine-regulated POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad comple... more NAC1 is a cocaine-regulated POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) protein. NAC1 is increased by cocaine selectively in the nucleus accumbens, a CNS region important for drug addiction. NAC1's role in the cell, however, is not known. Each of the two NAC1 isoforms, sNAC1 (short NAC1) and lNAC1 (long NAC1), may serve as corepressors for other POZ/BTB proteins. This study investigated whether sNAC1 and lNAC1 demonstrated proteinprotein interactions with other corepressors. Histone deacetylase (HDAC) inhibition reversed sNAC1 and lNAC1 repression of Gal4 luciferase, but only in neuronal-like cultures. Because these inhibitors do not distinguish among histone deacetylases, two histone deacetylases were selected for further study. HDAC 3 and 4 both demonstrated protein-protein interactions with sNAC1 and lNAC1. This was shown using coimmunoprecipitations, glutathione-S-transferase (GST) pulldowns and mammalian two-hybrids. Importantly, either the POZ domain or NAC1 without the POZ domain can bind these two HDACs. Other corepressors, specifically NCoR (nuclear receptor corepressor), SMRT (silencing mediator for retinoid and thyroid hormone receptor) and mSin3a, do not exhibit protein-protein interactions with sNAC1 and lNAC1. None showed protein-protein interactions in GST pulldowns or mammalian two-hybrids. Taken together, the results of these experiments indicate sNAC1 and lNAC1 recruit histone deacetylases for transcriptional repression, further enhancing POZ/BTB protein mediated repression.

Research paper thumbnail of Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin

Carcinogenesis, 1995

We explored the regulation of epidermal growth factor (EGF)-mediated activation of EGF receptor (... more We explored the regulation of epidermal growth factor (EGF)-mediated activation of EGF receptor (EGF-R) phosphorylation by curcumin (diferuloyl-methane), a recently identified kinase inhibitor, in cultured NIH 3T3 cells expressing human EGF-R. Treatment of cells with a saturating concentration of EGF for 5-15 min induced increased EGF-R tyrosine phosphorylation by 4- to 11-fold and this was inhibited in a dose- and time-dependent manner by up to 90% by curcumin, which also inhibited the growth of EGF-stimulated cells. There was no effect of curcumin treatment on the amount of surface expression of labeled EGF-R and inhibition of EGF-mediated tyrosine phosphorylation of EGF-R by curcumin was mediated by a reversible mechanism. In addition, curcumin also inhibited EGF-induced, but not bradykinin-induced, calcium release. These findings demonstrate that curcumin is a potent inhibitor of a growth stimulatory pathway, the ligand-induced activation of EGF-R, and may potentially be useful in developing anti-proliferative strategies to control tumor cell growth.

Research paper thumbnail of Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells

PubMed, 1997

Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveo... more Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveolar cells secrete surfactant in response to such secretagogs as terbutaline, calcium (Ca) ionophores (e.g., ionomycin [Io]), and adenosine triphosphate (ATP). A cell membrane receptor for SP-A, one of the surfactant proteins, regulates secretion by negative feedback. We used quantitative fluorescence microscopy to study the effects of SP-A on alterations in cytosolic Ca2+ ([Ca2+]i) elicited by surfactant secretagogs. Freshly isolated type II cells were loaded with Fura-2, then treated with secretagog, in the presence or absence of SP-A. Io and ATP produced biphasic increases in cytosol [Ca2+]i, reflecting first Ca2+ release from intracellular stores, and then influx through the cell membrane. Thapsigargin (TG) and Io directly initiate Ca2+ release; ATP elicits Ca2+ release via receptor-mediated mechanisms. Ca2+ release causes cell membrane Ca channels to open by as yet poorly understood mechanisms. Io itself acts as an additional Ca2+ channel. SP-A blocks much of the Ca2+ release and some of the Ca2+ influx elicited by these secretagogs. Antibody against SP-A receptor restores secretagog-induced Ca2+ fluxes from inhibition by SP-A, confirming that the inhibitory activity of SP-A is mediated through its receptor. Type II cells incubated in Ca2+-free medium plus SP-A show diminished Ca2+ release responses to TG or ATP, suggesting that the action of SP-A to prevent secretagog initiated increases in [Ca2+]i may reflect its ability to block Ca2+ release from cytoplasmic Ca stores. The feedback inhibition of surfactant secretion by SP-A may, correspondingly, be a manifestation of this effect. Because recent work suggests that TGF-beta also inhibits Ca2+ fluxes, SP-A and TGF-beta could be representative of a group of physiologic regulators that act by modulating intracellular Ca signaling.

Research paper thumbnail of Activity-dependent subcellular localization of NAC1

European Journal of Neuroscience, Jul 21, 2005

The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rat... more The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain.

Research paper thumbnail of Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes

Nature metabolism, Sep 7, 2020

Nature MetabolisM Extended Data Fig. 6 | Based on human-into-mouse islet transplant model, immuno... more Nature MetabolisM Extended Data Fig. 6 | Based on human-into-mouse islet transplant model, immunohistochemical profiling of islets ± IVM for markers of angiogenesis (VeGF), anti-apoptosis (Bcl-2, GLP-1) and endothelial cells (VWF) highlights the increased intensity and stained area of these epitopes in the IVM + group. The proportion and intensity of staining, quantified by QuPath using five automated regions of interest (ROIs) per sectioned image, is plotted below each section (* denotes p < 0.05 and ** denotes p < 5x10-5 based on the one-way Kruskal-Wallis H Test; arrow in the GLP-1 IVMsection denotes staining artifact). The particular significant p-values were-VEGF at POD 1 (p=0.009), Bcl-2 at POD1 (p=7x10-8) and POD10 (p=10-9), GLP-1 at POD1 (p=10-8) and POD 10 (p=4x10-9) and VWF at POD 10 (p=2x10-4).

Research paper thumbnail of Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium

Science Translational Medicine, May 22, 2019

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells with... more The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit + cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardio-pulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial †

Research paper thumbnail of Activation of surfactant protein-B transcription: Signaling through the SP-A receptor utilizing the PI3 kinase pathway

Journal of Cellular Physiology, 2000

This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and t... more This study describes receptor-activated signaling initiated by surfactant protein-A (SP-A), and the means by which it activates transcription of surfactant protein-B. Pulmonary surfactant is a mixture of lipids and associated proteins produced by type II pneumocytes. Interaction of SP-A with its cognate receptor (SPAR) on type II cells is involved in regulating surfactant secretion. This interaction also increases transcription of surfactant proteins and several other genes. To study SP-A cytokine activity, we used as a model surfactant-protein (SP-B) transcription, the activators of which have been characterized. HNF-3 and TTF-1 transcription factors are known to stimulate SP-B transcription. SP-A caused increased phosphorylation and nuclear localization of both. Corresponding increases in protein binding to the SP-B promoter were demonstrated by gel shift analysis. SP-A increased protein binding to HNF-3 and TTF-1 consensus recognition elements. Footprinting analysis indicated that SP-A-induced protein binding to SP-B promoter was greater in amount, but not different in location, from that seen in control cells, which normally transcribe SP-B. SP-A caused transient increases in PI3 kinase localization at the plasma membrane, and SP-A signaling to elicit increased SP-B transcription was blocked by LY294002, an inhibitor of PI3 kinase. Therefore, SP-A signals through PI3 kinase to increase SP-B transcription in type II pneumocytes by enhancing TTF-1 and HNF-3 activation of the SP-B promoter. SP-A activation of this signaling pathway, which affects many cellular functions and has not previously been implicated in type II cell transcriptional activity, has profound import for understanding type II cell biology.

Research paper thumbnail of SP-A as a cytokine: Surfactant protein-A-regulated transcription of surfactant proteins and other genes

Journal of Cellular Physiology, Mar 1, 1999

Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alv... more Pulmonary surfactant is a mixture of phospholipids and surfactant-associated proteins made by alveolar type II cells that is necessary for normal lung function. Surfactant secretion and reuptake by type II cells are regulated in part by interaction of surfactant protein-A (SP-A) with a specific receptor (SPAR) on type 11 cells. Several chemicals and hormones affect both surfactant secretion and also surfactant gene expression, but consequences of SP-A-SPAR interaction beyond regulating surfactant secretion and reuptake are unknown. Accordingly, we studied the effects of SP-A on surfactant protein gene transcription, mRNA levels, and transcript stability. SP-A elicited new transcription of surfactant proteins SP-A, SP-B, and SP-C and SPAR and c-Jun but had no effect on beta-actin or c-fos transcription. Antibody against SP-A receptor blocked SP-A-induced transcription, confirming that these actions of SP-A were receptor-mediated. SP-A effects on overall transcript levels were more complex. However, SP-A, SP-B, and SP-C mRNA levels doubled in SP-A-treated cells compared to controls. SP-A is known to stabilize surfactant, control its secretion and reuptake by type II cells, and augment host antimicrobial defenses. These data indicate that SP-A also acts as an autocrine cytokine: it binds its receptor and specifically regulates transcription of surfactant proteins and other genes.

Research paper thumbnail of NAC-1 Is a Brain POZ/BTB Protein That Can Prevent Cocaine-Induced Sensitization in the Rat

The Journal of Neuroscience, Aug 15, 2000

Levels of the mRNA NAC-1 are increased in the rat forebrain weeks after cocaine exposure. This lo... more Levels of the mRNA NAC-1 are increased in the rat forebrain weeks after cocaine exposure. This long-term neuroadaptation occurs during the expression of behavioral sensitization, a model of psychostimulant-induced paranoia. NAC-1, the protein encoded by this cocaine-regulated mRNA, contains a Pox virus and zinc finger/bric-a-brac tramtrack broad complex (POZ/BTB) motif, which mediates interactions among several transcriptional regulators. The present studies demonstrate that NAC-1 acts as a transcription factor. NAC-1 was localized to the nucleus of neurons in the brain. Transfection of NAC-1 in cell culture repressed transcription of a reporter gene. NAC-1 was also able to affect the actions of other POZ/BTB proteins in mammalian two-hybrid studies; these interactions required the presence of the POZ/BTB domain. However, NAC-1 appears to be a unique POZ/BTB transcriptional regulator because it does not contain any zinc finger regions found in these other DNA-binding proteins. Adenoviral-mediated overexpression of NAC-1 protein in the rat nucleus accumbens prevented the development but not the expression of behavioral sensitization produced by repeated administration of cocaine. Thus, NAC-1 may modify the longterm behaviors of psychostimulant abuse by regulating gene transcription in the mammalian brain.

Research paper thumbnail of 905: Release of placental exosomes is reduced in spontaneous preterm births

American Journal of Obstetrics and Gynecology, 2020

cannabinoid receptor interacting protein (CRIP) in laboring and non-laboring patients. STUDY DESI... more cannabinoid receptor interacting protein (CRIP) in laboring and non-laboring patients. STUDY DESIGN: Myometrial and placental tissue samples were collected in laboring and non-laboring women undergoing cesarean delivery. Tissue samples were rinsed and frozen at-80 C immediately after delivery. CB1 receptor and CRIP presence and quantification were evaluated using western blot and reverse transcription quantitative polymerase chain reaction. RESULTS: 20 low-risk patients were enrolled. The median gestational age was 38 weeks, 5 days in the labor group (n¼9) and 39 weeks in the non-labor group (n¼11). CB1 receptor presence was identified in myometrial and placental samples. Myometrial and placental tissue quantification showed no significant difference in CB1 receptor expression for all labor compared to non-labor patients (p¼0.4063) or in term spontaneous labor compared to non-labor patients (p¼0.7967). CRIP was identified with no difference in gene expression found in all myometrial (p¼0.2179) or placental (p¼0.7620) labor compared to non-labor tissues. Evaluation of term spontaneous labor compared to term non-labor patients showed significantly less CRIP gene expression in the myometrium of laboring patients (p¼0.0060). CONCLUSION: CB1 receptor and CRIP are present in human myometrial and placental tissues and may play a role in parturition. This role appears to be mediated by the cannabinoid receptor interacting protein.

Research paper thumbnail of Mechanism of interferon action: in vivo activation of 91 kDa transcription factor

PubMed, Sep 1, 1996

Interferons are a family of secreted polypeptides with distinct biological effects. These effects... more Interferons are a family of secreted polypeptides with distinct biological effects. These effects include the regulation of expression of specific cellular genes, antiviral properties, and inhibition of cell growth and proliferation. We investigated the effect of sodium orthovanadate (vanadate), an inhibitor of protein-phosphotyrosine phosphatases, on early biochemical events associated with the stimulation of transcription factor p91 activation by interferon-alpha (IFN alpha) in live human cells. We report that the treatment of cells with vanadate selectively potentiated (2-3 fold) the levels of IFN-stimulated tyrosine phosphorylation of p91 (and not of p113) as compared to the levels of p91 activated by IFN alone and that this was associated with the increased accumulation of phosphorylated p91 in the nucleus, and the activation of protein tyrosine kinases that phosphorylate p91. These results suggest the possible involvement of a vanadate sensitive protein-tyrosine phosphatase(s) in the deactivation of phosphorylated p91 in live human cells.

Research paper thumbnail of Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

Thrombosis and Haemostasis, 1994

Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in ra... more Recombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodinated and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivatized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.

Research paper thumbnail of Unlocking the Potential of Induced Pluripotent Stem Cells for Wound Healing: The Next Frontier of Regenerative Medicine

Advances in wound care, Nov 1, 2022

SIGNIFICANCE Non-healing wounds are a significant burden for the healthcare system all over the w... more SIGNIFICANCE Non-healing wounds are a significant burden for the healthcare system all over the world. Existing treatment options are not enough to promote healing, highlighting the urgent need for improved therapies. In addition, the current advancements in tissue-engineered skin constructs and stem cell-based therapies are facing significant hurdles due to the absence of a renewable source of functional cells. Recent Advances: Induced pluripotent stem cell technology (iPSC) is emerging as a novel tool to develop the next generation of personalized medicine for the treatment of chronic wounds. The iPSC provides unlimited access to various skin cells to generate complex personalized three-dimensional (3D) skin constructs for disease modeling and autologous grafts. Furthermore, the iPSC-based therapies can target distinct wound healing phases and have shown accelerating wound closure by enhancing angiogenesis, cell migration, tissue regeneration, and modulating inflammation. CRITICAL ISSUES Since the last decade iPSC has been revolutionizing the field of wound healing and skin tissue engineering. Despite the current progress, safety and heterogeneity among iPSC lines are still major hurdles in addition to the lack of large animal studies. These challenges need to be addressed before translating an iPSC-based therapy to the clinic. FUTURE DIRECTIONS Future considerations should be given to performing large animal studies to check the safety and efficiency of iPSC-based therapy in a wound healing setup. Furthermore, strategies should be developed to overcome variation between hiPSC lines, develop an efficient manufacturing process for iPSC-derived products, and generate complex skin constructs with vasculature and skin appendages.

Research paper thumbnail of Activity-dependent subcellular localization of NAC1

European Journal of Neuroscience, 2005

The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rat... more The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain.

Research paper thumbnail of Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection

American Journal of Transplantation

Research paper thumbnail of Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

Thrombosis and Haemostasis, 1994

SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activit... more SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodina- ted and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivitized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening tha...

Research paper thumbnail of Donor Lung Specific Exosome Profiles for Noninvasive Monitoring of Acute Rejection in a Rat Orthotopic Left Lung Transplant Model

The Journal of Heart and Lung Transplantation, 2018

Purpose: There is a critical need for development of biomarkers to monitor for lung transplant re... more Purpose: There is a critical need for development of biomarkers to monitor for lung transplant rejection. We investigated the biomarker potential of circulating donor lung specific exosome profiles for time sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. Methods: Left lung from Wistar transgenic rat expressing exosome marker human specific CD63-GFP was transplanted into Lewis recipient across full major histocompatibility complex mismatch (n= 12). Recipient blood was collected daily (days 0 to 8) and plasma was processed for exosome isolation by size exclusion chromatography and ultracentrifugation. Donor lung specific exosomes were profiled using anti-human CD63 antibody quantum dot on the nanoparticle detector. Fluorescence microscopy for GFP was performed to understand donor lung exosome trafficking during the acute rejection process.

Research paper thumbnail of Intramyocardial cell-based therapy during bidirectional cavopulmonary anastomosis for hypoplastic left heart syndrome: The ELPIS phase I trial

ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstructi... more ABSTRACTBackgroundHypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction for the right ventricle (RV) to provide systemic circulation. This leads to substantially increased loads on the RV, wall stress, maladaptive remodeling and dysfunction, which in turn can increase risk of death or transplantation.ObjectivesWe conducted a phase I multicenter trial to assess safety and feasibility of intra-operative MSC injection in HLHS patients to boost RV performance in the systemic position.MethodsAllogeneic MSCs were directly administered by intramyocardial injections during the second stage palliative operation. The primary endpoint was safety.ResultsTen patients received intramyocardial injections of allogeneic MSCs (Lomecel-B). No patients experienced major adverse cardiac events (MACE). All subjects were alive and transplant-free at 1 year following, and experienced growth comparable to healthy control historical data. Cardiac magnetic resonance imaging (CMR) r...