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1.
Biochem Biophys Res Commun ; 614: 207-212, 2022 Jul 23.
Article in English | MEDLINE | ID: covidwho-1814155

ABSTRACT

Simple, highly sensitive detection technologies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for the effective implementation of public health policies. We used the systematic evolution of ligands by exponential enrichment with a modified DNA library, including a base-appended base (uracil with a guanine base at its fifth position), to create an aptamer with a high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. The aptamer had a dissociation constant of 1.2 and < 1 nM for the RBD and spike trimer, respectively. Furthermore, enzyme-linked aptamer assays confirmed that the aptamer binds to isolated authentic SARS-CoV-2 wild-type and B.1.617.2 (delta variant). The binding signal was larger that of commercially available anti-SARS-CoV-2 RBD antibody. Thus, this aptamer as a sensing element will enable the highly sensitive detection of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , DNA/metabolism , Humans , Oligonucleotides/metabolism , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
2.
Int J Mol Sci ; 23(9)2022 Apr 27.
Article in English | MEDLINE | ID: covidwho-1809941

ABSTRACT

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO-DNA and Histone-DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.


Subject(s)
COVID-19 , Extracellular Traps , Thrombosis , Anti-Inflammatory Agents/pharmacology , COVID-19/drug therapy , Cytokines/metabolism , DNA/metabolism , Enoxaparin/pharmacology , Enoxaparin/therapeutic use , Extracellular Traps/metabolism , Humans , Neutrophils/metabolism , Thrombosis/drug therapy , Thrombosis/metabolism
3.
Int J Mol Sci ; 22(21)2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1745034

ABSTRACT

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.


Subject(s)
DNA/chemistry , DNA/metabolism , Dendrimers/chemistry , Phenols/chemistry , Sulfides/chemistry , Animals , Male , Molecular Conformation , Salmon , Spermatozoa/metabolism
4.
Int J Mol Sci ; 23(6)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1742489

ABSTRACT

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.


Subject(s)
DNA/genetics , Gene Transfer Techniques , Methane/chemistry , Pyridinium Compounds/chemistry , Surface-Active Agents/chemistry , Transfection/methods , A549 Cells , Cell Survival , DNA/chemistry , DNA/metabolism , Genetic Therapy/methods , Halogenation , Humans , Hydrogenation , Methane/metabolism , Microscopy, Atomic Force , Molecular Structure , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Pyridinium Compounds/metabolism , Reproducibility of Results , Surface-Active Agents/metabolism
5.
Int J Biol Macromol ; 203: 466-480, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1630871

ABSTRACT

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.


Subject(s)
COVID-19/virology , Nucleic Acids/metabolism , Nucleocapsid Proteins/metabolism , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , Binding Sites , DNA/chemistry , DNA/metabolism , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Humans , Hydrogen Bonding , Models, Molecular , Nucleic Acids/chemistry , Nucleocapsid Proteins/chemistry , Protein Binding , RNA/chemistry , RNA/metabolism , Spectrum Analysis , Structure-Activity Relationship
6.
Biologicals ; 75: 12-15, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1616379

ABSTRACT

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.


Subject(s)
DNA/genetics , Epidermal Growth Factor/chemistry , Gene Expression , Peptides , Plasmids/genetics , Plasmids/metabolism , Protein Domains , Animals , COVID-19 Vaccines , Cell Membrane/metabolism , DNA/metabolism , Genes, Reporter , Green Fluorescent Proteins/genetics , Mice , Muscle, Skeletal , Vaccines, DNA/genetics , Vaccines, DNA/metabolism
7.
PLoS One ; 16(12): e0246916, 2021.
Article in English | MEDLINE | ID: covidwho-1546847

ABSTRACT

The COVID-19 pandemic has reintroduced questions regarding the potential risk of SARS-CoV-2 exposure amongst passengers on an aircraft. Quantifying risk with computational fluid dynamics models or contact tracing methods alone is challenging, as experimental results for inflight biological aerosols is lacking. Using fluorescent aerosol tracers and real time optical sensors, coupled with DNA-tagged tracers for aerosol deposition, we executed ground and inflight testing on Boeing 767 and 777 airframes. Analysis here represents tracer particles released from a simulated infected passenger, in multiple rows and seats, to determine the exposure risk via penetration into breathing zones in that row and numerous rows ahead and behind the index case. We present here conclusions from 118 releases of fluorescent tracer particles, with 40+ Instantaneous Biological Analyzer and Collector sensors placed in passenger breathing zones for real-time measurement of simulated virus particle penetration. Results from both airframes showed a minimum reduction of 99.54% of 1 µm aerosols from the index source to the breathing zone of a typical passenger seated directly next to the source. An average 99.97 to 99.98% reduction was measured for the breathing zones tested in the 767 and 777, respectively. Contamination of surfaces from aerosol sources was minimal, and DNA-tagged 3 µm tracer aerosol collection techniques agreed with fluorescent methodologies.


Subject(s)
Aircraft , Computer Simulation , Fluorescent Dyes/chemistry , /chemistry , COVID-19/pathology , COVID-19/prevention & control , COVID-19/virology , DNA/chemistry , DNA/metabolism , Humans , Masks , Microspheres , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
8.
Biochem J ; 478(14): 2789-2791, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1526112

ABSTRACT

Post-translational modifications (PTMs) on histone proteins are known as epigenetic marks that demarcate the status of chromatin. These modifications are 'read' by specific reader proteins, which in turn recruit additional factors to modulate chromatin accessibility and the activity of the underlying DNA. Accumulating evidence suggests that these modifications are not restricted solely to histones, many non-histone proteins may function in a similar way through mimicking the histones. In this commentary, we briefly discuss a systematic study of the discovery of histone H3 N-terminal mimicry proteins (H3TMs), and their implications in chromatin regulation and drug discoveries.


Subject(s)
Chromatin/metabolism , DNA/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Animals , Chromatin/genetics , Chromatin Assembly and Disassembly , DNA/genetics , Humans , Lysine/metabolism , Methylation , Models, Biological
9.
Front Immunol ; 12: 714833, 2021.
Article in English | MEDLINE | ID: covidwho-1506100

ABSTRACT

Background: The most severe cases of Coronavirus-Disease-2019 (COVID-19) develop into Acute Respiratory Distress Syndrome (ARDS). It has been proposed that oxygenation may be inhibited by extracellular deoxyribonucleic acid (DNA) in the form of neutrophil extracellular traps (NETs). Dornase alfa (Pulmozyme, Genentech) is recombinant human deoxyribonuclease I that acts as a mucolytic by cleaving and degrading extracellular DNA. We performed a pilot study to evaluate the effects of dornase alfa in patients with ARDS secondary to COVID-19. Methods: We performed a pilot, non-randomized, case-controlled clinical trial of inhaled dornase for patients who developed ARDS secondary to COVID-19 pneumonia. Results: Improvement in arterial oxygen saturation to inhaled fraction of oxygen ratio (PaO2/FiO2) was noted in the treatment group compared to control at day 2 (95% CI, 2.96 to 95.66, P-value = 0.038), as well as in static lung compliance at days 3 through 5 (95% CI, 4.8 to 19.1 mL/cmH2O, 2.7 to 16.5 mL/cmH2O, and 5.3 to 19.2 mL/cmH2O, respectively). These effects were not sustained at 14 days. A reduction in bronchoalveolar lavage fluid (BALF) myeloperoxidase-DNA (DNA : MPO) complexes (95% CI, -14.7 to -1.32, P-value = 0.01) was observed after therapy with dornase alfa. Conclusion: Treatment with dornase alfa was associated with improved oxygenation and decreased DNA : MPO complexes in BALF. The positive effects, however, were limited to the time of drug delivery. These data suggest that degradation of extracellular DNA associated with NETs or other structures by inhaled dornase alfa can be beneficial. We propose a more extensive clinical trial is warranted. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT04402970.


Subject(s)
COVID-19/drug therapy , Deoxyribonuclease I/therapeutic use , Respiratory Distress Syndrome/drug therapy , SARS-CoV-2/physiology , Administration, Inhalation , Adult , Aged , Aged, 80 and over , Case-Control Studies , DNA/metabolism , Extracellular Traps/metabolism , Female , Humans , Male , Middle Aged , Oxygen Consumption/drug effects , Peroxidase/metabolism , Pilot Projects , Recombinant Proteins/therapeutic use , Young Adult
10.
Biomolecules ; 11(11)2021 10 20.
Article in English | MEDLINE | ID: covidwho-1480577

ABSTRACT

SARS-CoV-2 contains certain molecules that are related to the presence of immunothrombosis. Here, we review the pathogen and damage-associated molecular patterns. We also study the imbalance of different molecules participating in immunothrombosis, such as tissue factor, factors of the contact system, histones, and the role of cells, such as endothelial cells, platelets, and neutrophil extracellular traps. Regarding the pathogenetic mechanism, we discuss clinical trials, case-control studies, comparative and translational studies, and observational studies of regulatory or inhibitory molecules, more specifically, extracellular DNA and RNA, histones, sensors for RNA and DNA, as well as heparin and heparinoids. Overall, it appears that a network of cells and molecules identified in this axis is simultaneously but differentially affecting patients at different stages of COVID-19, and this is characterized by endothelial damage, microthrombosis, and inflammation.


Subject(s)
Alarmins , COVID-19/virology , SARS-CoV-2 , Thrombosis/virology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Blood Coagulation , Blood Platelets/virology , COVID-19/complications , DNA/metabolism , Extracellular Traps , Heparin/metabolism , Histones/metabolism , Humans , Mice , Neuropilin-1/metabolism , RNA/metabolism , Signal Transduction , Thrombin/metabolism , Thromboplastin/metabolism , Thrombosis/complications
11.
Inorg Chem ; 59(23): 17109-17122, 2020 Dec 07.
Article in English | MEDLINE | ID: covidwho-1387106

ABSTRACT

Metal complexes have numerous applications in the current era, particularly in the field of pharmaceutical chemistry and catalysis. A novel synthetic approach for the same is always a beneficial addition to the literature. Henceforth, for the first time, we report the formation of three new Pd(II) complexes through the Michael addition pathway. Three chromone-based thiosemicarbazone ligands (SVSL1-SVSL3) and Pd(II) complexes (1-3) were synthesized and characterized by analytical and spectroscopic tools. The Michael addition pathway for the formation of complexes was confirmed by spectroscopic studies. Distorted square planar structure of complex 2 was confirmed by single-crystal X-ray diffraction. Complexes 1-3 were subjected to DNA- and BSA-binding studies. The complex with cyclohexyl substituent on the terminal N of thiosemicarbazone (3) showed the highest binding efficacy toward these biomolecules, which was further understood through molecular docking studies. The anticancer potential of these complexes was studied preliminarily by using MTT assay in cancer and normal cell lines along with the benchmark drugs (cisplatin, carboplatin, and gemcitabine). It was found that complex 3 was highly toxic toward MDA-MB-231 and AsPC-1 cancer cells with IC50 values of 0.5 and 0.9 µM, respectively, and was more efficient than the standard drugs. The programmed cell death mechanism of the complexes in MDA-MB-231 cancer cells was confirmed. Furthermore, the complexes induced apoptosis via ROS-mediated mitochondrial signaling pathway. Conveniently, all the complexes showed less toxicity (≥50 µM) against MCF-10a normal cell line. Molecular docking studies were performed with VEGFR2, EGFR, and SARS-CoV-2 main protease to illustrate the binding efficiency of the complexes with these receptors. To our surprise, binding potential of the complexes with SARS-CoV-2 main protease was higher than that with chloroquine and hydroxychloroquine.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Coordination Complexes/pharmacology , Mitochondria/drug effects , Reactive Oxygen Species/metabolism , SARS-CoV-2/enzymology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Chromones/chemical synthesis , Chromones/metabolism , Chromones/pharmacology , Coordination Complexes/chemical synthesis , Coordination Complexes/metabolism , Coronavirus 3C Proteases/metabolism , DNA/metabolism , Drug Screening Assays, Antitumor , ErbB Receptors/metabolism , Humans , Intercalating Agents/chemical synthesis , Intercalating Agents/metabolism , Intercalating Agents/pharmacology , Ligands , Molecular Docking Simulation , Palladium/chemistry , Protein Binding , Thiosemicarbazones/chemical synthesis , Thiosemicarbazones/metabolism , Thiosemicarbazones/pharmacology , Vascular Endothelial Growth Factor Receptor-2/metabolism
12.
Am J Med Sci ; 361(5): 585-590, 2021 05.
Article in English | MEDLINE | ID: covidwho-1085595

ABSTRACT

BACKGROUND: Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) oxidative damage is associated with mortality of patients with different diseases. However, there are no data about DNA and RNA oxidative damage from coronavirus disease 2019 (COVID-19) patients. Thus, the objective of this study was to explore DNA and RNA oxidative damage in surviving and non-surviving COVID-19 patients. MATERIALS AND METHODS: Eight Intensive Care Units from 6 hospitals in the Canary Islands (Spain) participated in this prospective and observational study. We recorded the serum levels at ICU admission of the three guanine oxidized species (OGS) because guanine is the nucleobase that forms the DNA and RNA most prone to oxidation. Survival at 30 days was our end-point study. RESULTS: Non-surviving (n = 11) compared to surviving patients (n = 42) had higher APACHE-II (p < 0.001), SOFA (p = 0.004) and serum OGS levels (p = 0.001). In logistic regression analyses an association between serum OGS levels and 30-day mortality after controlling for SOFA (OR=2.601; 95% CI=1.305-5.182; p = 0.007) or APACHE-II (OR=2.493; 95% CI=1.274-4.879; p = 0.008) was found. The area under curve (AUC) for mortality prediction by serum OGS levels was 83% (95% CI=70-92%; p < 0.001), by APACHE II was 85% (95% CI=75-96%; p < 0.001), and by SOFA was 80% (95% CI=66-94%; p < 0.001). No significant differences were found in the AUC between serum OGS levels and SOFA (p = 0.91), and serum OGS levels and APACHE-II (p = 0.64). CONCLUSIONS: To our knowledge, this is the first study reporting on oxidative DNA and RNA damage in COVID-19 patients, and the main new finding was that serum OGS concentration was associated with mortality.


Subject(s)
COVID-19 , DNA Damage , DNA/metabolism , RNA/metabolism , SARS-CoV-2/metabolism , Aged , COVID-19/metabolism , COVID-19/mortality , Disease-Free Survival , Female , Humans , Male , Middle Aged , Oxidation-Reduction , Spain/epidemiology , Survival Rate
13.
Sci Rep ; 10(1): 4481, 2020 03 11.
Article in English | MEDLINE | ID: covidwho-7753

ABSTRACT

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5' single-stranded tail in a 5' to 3' direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5'-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.


Subject(s)
Adenosine Triphosphate/metabolism , Methyltransferases/metabolism , RNA Helicases/metabolism , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , SARS Virus/enzymology , Viral Nonstructural Proteins/metabolism , DNA/metabolism , DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , Hydrolysis , Kinetics , Protein Binding , RNA-Binding Proteins/metabolism , Substrate Specificity , Virus Replication/physiology
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