<p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (I... more <p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. The lane marked NIC represents IP of U937/Nef-EYFP lysates with an irrelevant antibody. (B) U1 and J1.1 cells were treated with DMSO (–) or PMA (+) and cell lysates were either immunoblotted for p24 and Nef (i, ii), or IP/IB with anti-Nef and anti-Ago2 (iii, iv), as described in Materials and Methods. U937 and Jurkat cells served as background controls and antibody heavy chain as loading control. (C) Lysates were prepared from the U937 stable cell lines using Buffer D as described in Materials and Methods. From this, 150 µg lysate was passed through a pre-equilibrated Sephacryl S200HR column with a 3 ml bed volume. The column was eluted and fractions of 3 drops (∼120 µl) were collected, which were then precipitated with acetone, separated by SDS-PAGE and western blotting was done with Ago2 and GFP antibodies. (D) Lysates from activated U1 cells were prepared and fractionated as in (C) followed by SDS-PAGE and western blotting with Ago2 and Nef antibodies. The profiles shown are representative of three separate experiments.</p
<p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los A... more <p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los Alamos HIV database were analyzed. Regions encompassing the two conserved GW motifs (bold and underline) at positions 12–13 and 140–141 are shown for consensus and ancestral (.anc) sequences for various HIV-1 M group clades (A–H), and consensus sequences for various recombinant circulating forms. (B) HEK293T cells were transiently transfected to express wild type or GW mutant Nef proteins. Cell lysates were precipitated with anti-Nef and blotted with anti-Ago2. Nef and Actin were expression and loading controls, respectively. (C) HEK293T cells were transfected with either pNL4-3 or pNL4-3ΔNef; cells were also co-transfected with pNL4-3ΔNef and expression vectors for either the wild type or GW mutant Nef proteins. After 36 hr the culture supernatants were quantified for p24. Purified viruses equivalent to 100ng p24 were also used to infect 0.5 million 1G5 Jurkat indicator cells, and 48 hr later the cells were harvested, lysed and luciferase assay was performed as described in Methods. Error bars represent mean ± SD from three independent experiments, and p-values calculated using the Student’s <i>t</i>-test were as follows: * 0.002; # 0.023.</p
<p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established a... more <p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established as described in Materials and Methods. (A) Western blot for expression of EYFP (lanes Y6-A to -D) and Nef-EYFP (lanes NY-19 and -20) in selected U937 stable clones. (B) Flow cytometric analysis of U937/Nef-EYFP and U937/EYFP cells for surface expression of the indicated proteins. (C) The U937/Nef-EYFP cells were (i) cultured with 5 µM NRhPE, or (ii) stained with anti-Ago2 as described in Materials and Methods. U1 cells were activated with PMA and labeled with anti-Nef and either (iii) anti-Ago2 or (iv) anti-P bodies as described in Materials and Methods. The white arrowheads in the merged image represent colocalization points. The images are representative of three independent experiments. The boxed regions are expanded. (D) Correlation analysis of the colocalization of Nef with Ago2 in (i) U937/Nef-EYFP cells and (ii) activated U1 cells showing Pearson’s coefficient (PC) and Manders coefficients (M1, M2). The coefficients represent an average of three independent images, each consisting of at least 10 cells, calculated using the JACoP plugin within the Image J software. M1 represents the fraction of Ago2-red overlapping with Nef-green and M2 represents the fraction of Nef-green overlapping with Ago2- red. Bars represent ± SD.</p
<p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Ma... more <p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Materials and Methods. From this, 25 µg of lysate was incubated with <sup>32</sup>P-labeled duplex siRNA (without or with 50-fold molar excess of unlabeled siRNA) in binding buffer for 30 min at 4°C. The complexes were separated on a 6% non-denaturing polyacrylamide gel. (B) The gel shift assay was set up as in (A) except that lysates immunodepleted for either Ago2 or Dicer were also included. The positions of the mobility shifted complexes and free probe are indicated. (C) Slicer activity was assayed using immunoprecipitated Ago2 from U937/Nef-EYFP or U937/EYFP cell lysates and a <sup>32</sup>P-labeled <i>in vitro</i> transcribed let7a RNA, as described in Materials and Methods. The positions of full-length RNA and the two sliced products are shown. The 59 nt marker oligonucleotide was run on the same gel, but a lower exposure is shown. (D) Densitometric analysis of autoradiograms from three independent experiments was carried out using Image J (version 1.4.1). The slicing activity in Nef-EYFP lysates is represented as a percentage of that in EYFP lysates.</p
Introduction: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellul... more Introduction: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins
Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remai... more Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remains untreated, it leads to high rate of morbidity and mortality, and a median survival of only 5–7 years. Clinically, PAH is defined as mean pulmonary arterial pressure >20 mmHg at rest with normal left atrial pressure. PAH is characterized by remodeling of the pulmonary vasculature due to pulmonary vascular endothelial and smooth cell proliferation. These events eventually reduce the pulmonary arterial compliance and thus causing increased pulmonary vascular resistance. If remains unmanaged, it will ultimately end up in right ventricular failure. However, the Food and Drug Administration (FDA) has approved drugs that target the endothelial pathway, the nitric oxide pathway, and the prostacyclin pathway. These therapeutic strategies, mainly inducing pulmonary vasodilation, however, have poor effect on the signaling pathways activated during the pathogenesis of PAH. This might be one of...
Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remai... more Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remains untreated, it leads to high rate of morbidity and mortality, and a median survival of only 5–7 years. Clinically, PAH is defined as mean pulmonary arterial pressure >20 mmHg at rest with normal left atrial pressure. PAH is characterized by remodeling of the pulmonary vasculature due to pulmonary vascular endothelial and smooth cell proliferation. These events eventually reduce the pulmonary arterial compliance and thus causing increased pulmonary vascular resistance. If remains unmanaged, it will ultimately end up in right ventricular failure. However, the Food and Drug Administration (FDA) has approved drugs that target the endothelial pathway, the nitric oxide pathway, and the prostacyclin pathway. These therapeutic strategies, mainly inducing pulmonary vasodilation, however, have poor effect on the signaling pathways activated during the pathogenesis of PAH. This might be one of the reasons why they are unable to reverse the pathology of the disease, and eventually patients stop responding to them. If medications fail, the only viable alternative will be lung transplantation to save a patient’s life. Evidences show the contribution of S. Heo · N. Ly · M. Shahid · M. Tauseef (*) Department of Pharmaceutical Sciences, Chicago State University College of Pharmacy, Chicago, IL, USA e-mail: [email protected] M. Aqil Freelance Medical Writer, American Medical Writers Association (AMWA), Rockville, MD, USA M. R. Siddiqui Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Z. Ahmad Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, USA # Springer Nature Singapore Pte Ltd. 2020 S. Rayees et al. (eds.), Chronic Lung Diseases, https://doi.org/10.1007/978-981-15-3734-9_1 1 heredity, inflammation, drugs, toxins, HIV infection, and hepatic diseases in the progression of PAH. Therefore, there is a need for potential therapeutics, which, via affecting the pathological signaling pathways, halt the disease process and eventually reverse the pulmonary remodeling in the patients. In this chapter, we discuss the emerging novel therapeutics and their mechanism of actions, which have advanced beyond preclinical research and are currently being investigated in the clinical cases of PAH.
Advances in Medical Diagnosis, Treatment, and Care
During inflammatory conditions, such as sepsis, myocardial infarction and acute respiratory distr... more During inflammatory conditions, such as sepsis, myocardial infarction and acute respiratory distress syndrome, endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications and ultimately leads to the multi-organ failure and death. Thus, to determine the molecular mechanisms underlying the opening and resolution phase of the gap formation, will provide an insight to better understand the pathology of the cardiovascular and pulmonary inflammatory disorders. In this chapter, we will discuss about how the signaling mechanisms activated by the known inflammatory molecules increase endothelial permeability.
The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression ... more The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes fro...
<p>Exosomes were purified from the spent culture media of the two cell lines as detailed in... more <p>Exosomes were purified from the spent culture media of the two cell lines as detailed in Materials and Methods. (A) Western blotting was done using 40 µg of cellular and exosomal lysates using antibodies that detect marker proteins for exosomes (Alix, Tsg101, CD81), mitochondria (VDAC), endoplasmic reticulum (Calnexin) and apoptotic bodies (Cytochrome C). GAPDH and Actin were used as loading controls and anti-GFP antibodies were used to detect Nef-EYFP and EYFP. (B) Cellular and exosomal lysates were subjected to SDS-PAGE and western blotting to detect GW182, Ago2 and Nef; GAPDH was used as a loading control.</p
<p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (I... more <p>(A) Lysates of U937/Nef-EYFP or U937/EYFP cells were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. The lane marked NIC represents IP of U937/Nef-EYFP lysates with an irrelevant antibody. (B) U1 and J1.1 cells were treated with DMSO (–) or PMA (+) and cell lysates were either immunoblotted for p24 and Nef (i, ii), or IP/IB with anti-Nef and anti-Ago2 (iii, iv), as described in Materials and Methods. U937 and Jurkat cells served as background controls and antibody heavy chain as loading control. (C) Lysates were prepared from the U937 stable cell lines using Buffer D as described in Materials and Methods. From this, 150 µg lysate was passed through a pre-equilibrated Sephacryl S200HR column with a 3 ml bed volume. The column was eluted and fractions of 3 drops (∼120 µl) were collected, which were then precipitated with acetone, separated by SDS-PAGE and western blotting was done with Ago2 and GFP antibodies. (D) Lysates from activated U1 cells were prepared and fractionated as in (C) followed by SDS-PAGE and western blotting with Ago2 and Nef antibodies. The profiles shown are representative of three separate experiments.</p
<p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los A... more <p>(A) Alignment of Nef sequences. All 2660 Nef amino acid sequences available in the Los Alamos HIV database were analyzed. Regions encompassing the two conserved GW motifs (bold and underline) at positions 12–13 and 140–141 are shown for consensus and ancestral (.anc) sequences for various HIV-1 M group clades (A–H), and consensus sequences for various recombinant circulating forms. (B) HEK293T cells were transiently transfected to express wild type or GW mutant Nef proteins. Cell lysates were precipitated with anti-Nef and blotted with anti-Ago2. Nef and Actin were expression and loading controls, respectively. (C) HEK293T cells were transfected with either pNL4-3 or pNL4-3ΔNef; cells were also co-transfected with pNL4-3ΔNef and expression vectors for either the wild type or GW mutant Nef proteins. After 36 hr the culture supernatants were quantified for p24. Purified viruses equivalent to 100ng p24 were also used to infect 0.5 million 1G5 Jurkat indicator cells, and 48 hr later the cells were harvested, lysed and luciferase assay was performed as described in Methods. Error bars represent mean ± SD from three independent experiments, and p-values calculated using the Student’s <i>t</i>-test were as follows: * 0.002; # 0.023.</p
<p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established a... more <p>U937 cells stably expressing either a Nef-EYFP fusion protein or EYFP were established as described in Materials and Methods. (A) Western blot for expression of EYFP (lanes Y6-A to -D) and Nef-EYFP (lanes NY-19 and -20) in selected U937 stable clones. (B) Flow cytometric analysis of U937/Nef-EYFP and U937/EYFP cells for surface expression of the indicated proteins. (C) The U937/Nef-EYFP cells were (i) cultured with 5 µM NRhPE, or (ii) stained with anti-Ago2 as described in Materials and Methods. U1 cells were activated with PMA and labeled with anti-Nef and either (iii) anti-Ago2 or (iv) anti-P bodies as described in Materials and Methods. The white arrowheads in the merged image represent colocalization points. The images are representative of three independent experiments. The boxed regions are expanded. (D) Correlation analysis of the colocalization of Nef with Ago2 in (i) U937/Nef-EYFP cells and (ii) activated U1 cells showing Pearson’s coefficient (PC) and Manders coefficients (M1, M2). The coefficients represent an average of three independent images, each consisting of at least 10 cells, calculated using the JACoP plugin within the Image J software. M1 represents the fraction of Ago2-red overlapping with Nef-green and M2 represents the fraction of Nef-green overlapping with Ago2- red. Bars represent ± SD.</p
<p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Ma... more <p>(A) Lysates from the U937 stable cell lines were prepared in Buffer D as described in Materials and Methods. From this, 25 µg of lysate was incubated with <sup>32</sup>P-labeled duplex siRNA (without or with 50-fold molar excess of unlabeled siRNA) in binding buffer for 30 min at 4°C. The complexes were separated on a 6% non-denaturing polyacrylamide gel. (B) The gel shift assay was set up as in (A) except that lysates immunodepleted for either Ago2 or Dicer were also included. The positions of the mobility shifted complexes and free probe are indicated. (C) Slicer activity was assayed using immunoprecipitated Ago2 from U937/Nef-EYFP or U937/EYFP cell lysates and a <sup>32</sup>P-labeled <i>in vitro</i> transcribed let7a RNA, as described in Materials and Methods. The positions of full-length RNA and the two sliced products are shown. The 59 nt marker oligonucleotide was run on the same gel, but a lower exposure is shown. (D) Densitometric analysis of autoradiograms from three independent experiments was carried out using Image J (version 1.4.1). The slicing activity in Nef-EYFP lysates is represented as a percentage of that in EYFP lysates.</p
Introduction: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellul... more Introduction: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins
Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remai... more Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remains untreated, it leads to high rate of morbidity and mortality, and a median survival of only 5–7 years. Clinically, PAH is defined as mean pulmonary arterial pressure >20 mmHg at rest with normal left atrial pressure. PAH is characterized by remodeling of the pulmonary vasculature due to pulmonary vascular endothelial and smooth cell proliferation. These events eventually reduce the pulmonary arterial compliance and thus causing increased pulmonary vascular resistance. If remains unmanaged, it will ultimately end up in right ventricular failure. However, the Food and Drug Administration (FDA) has approved drugs that target the endothelial pathway, the nitric oxide pathway, and the prostacyclin pathway. These therapeutic strategies, mainly inducing pulmonary vasodilation, however, have poor effect on the signaling pathways activated during the pathogenesis of PAH. This might be one of...
Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remai... more Pulmonary arterial hypertension (PAH) is a progressive disease with multiple etiologies. If remains untreated, it leads to high rate of morbidity and mortality, and a median survival of only 5–7 years. Clinically, PAH is defined as mean pulmonary arterial pressure >20 mmHg at rest with normal left atrial pressure. PAH is characterized by remodeling of the pulmonary vasculature due to pulmonary vascular endothelial and smooth cell proliferation. These events eventually reduce the pulmonary arterial compliance and thus causing increased pulmonary vascular resistance. If remains unmanaged, it will ultimately end up in right ventricular failure. However, the Food and Drug Administration (FDA) has approved drugs that target the endothelial pathway, the nitric oxide pathway, and the prostacyclin pathway. These therapeutic strategies, mainly inducing pulmonary vasodilation, however, have poor effect on the signaling pathways activated during the pathogenesis of PAH. This might be one of the reasons why they are unable to reverse the pathology of the disease, and eventually patients stop responding to them. If medications fail, the only viable alternative will be lung transplantation to save a patient’s life. Evidences show the contribution of S. Heo · N. Ly · M. Shahid · M. Tauseef (*) Department of Pharmaceutical Sciences, Chicago State University College of Pharmacy, Chicago, IL, USA e-mail: [email protected] M. Aqil Freelance Medical Writer, American Medical Writers Association (AMWA), Rockville, MD, USA M. R. Siddiqui Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Z. Ahmad Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, USA # Springer Nature Singapore Pte Ltd. 2020 S. Rayees et al. (eds.), Chronic Lung Diseases, https://doi.org/10.1007/978-981-15-3734-9_1 1 heredity, inflammation, drugs, toxins, HIV infection, and hepatic diseases in the progression of PAH. Therefore, there is a need for potential therapeutics, which, via affecting the pathological signaling pathways, halt the disease process and eventually reverse the pulmonary remodeling in the patients. In this chapter, we discuss the emerging novel therapeutics and their mechanism of actions, which have advanced beyond preclinical research and are currently being investigated in the clinical cases of PAH.
Advances in Medical Diagnosis, Treatment, and Care
During inflammatory conditions, such as sepsis, myocardial infarction and acute respiratory distr... more During inflammatory conditions, such as sepsis, myocardial infarction and acute respiratory distress syndrome, endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications and ultimately leads to the multi-organ failure and death. Thus, to determine the molecular mechanisms underlying the opening and resolution phase of the gap formation, will provide an insight to better understand the pathology of the cardiovascular and pulmonary inflammatory disorders. In this chapter, we will discuss about how the signaling mechanisms activated by the known inflammatory molecules increase endothelial permeability.
The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression ... more The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes fro...
<p>Exosomes were purified from the spent culture media of the two cell lines as detailed in... more <p>Exosomes were purified from the spent culture media of the two cell lines as detailed in Materials and Methods. (A) Western blotting was done using 40 µg of cellular and exosomal lysates using antibodies that detect marker proteins for exosomes (Alix, Tsg101, CD81), mitochondria (VDAC), endoplasmic reticulum (Calnexin) and apoptotic bodies (Cytochrome C). GAPDH and Actin were used as loading controls and anti-GFP antibodies were used to detect Nef-EYFP and EYFP. (B) Cellular and exosomal lysates were subjected to SDS-PAGE and western blotting to detect GW182, Ago2 and Nef; GAPDH was used as a loading control.</p
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