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Journal of Virology Sep 2021Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate...
Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a nearinfrared fluorescent protein (iRFP) upstream of . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication and reporter expression and . Both codon-optimized reporter viruses could be visualized during coinfection and reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.
Topics: Animals; CD4-Positive T-Lymphocytes; Dinucleoside Phosphates; Gene Expression; Genes, Reporter; HIV Infections; HIV-1; Humans; Luciferases; Luminescent Measurements; Luminescent Proteins; Mice; Optical Imaging; Viremia; Virus Replication; Whole Body Imaging
PubMed: 34232063
DOI: 10.1128/JVI.00449-21 -
Journal of Biomolecular Structure &... 2022Human stimulator of interferon genes (STING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic...
Human stimulator of interferon genes (STING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3'-3', 2'-2' or mixed 2'-3'-linkages) and exhibit different binding affinity towards STING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with STING protein. Our results revealed that CDN/STING interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in STING ligand binding cavity, especially in α-helices, the so-called lid region and α-tails. The ligand residence time in STING protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of STING protein. These findings may perhaps guide design of new compounds modulating STING activity.Communicated by Ramaswamy H. Sarma.
Topics: Humans; Molecular Dynamics Simulation; Ligands; Dinucleoside Phosphates; DNA; Oligonucleotides
PubMed: 34187319
DOI: 10.1080/07391102.2021.1942213 -
Physiological Reports Jun 2021Hypercholesterolemia and oxidative stress may lead to disturbances in the renal microvasculature in response to vasoactive agents, including P2 receptors (P2R) agonists....
Hypercholesterolemia and oxidative stress may lead to disturbances in the renal microvasculature in response to vasoactive agents, including P2 receptors (P2R) agonists. We investigated the renal microvascular response to diadenosine tetraphosphate (Ap A), an agonist of P2R, in diet-induced hypercholesteremic rats over 28 days, supplemented in the last 10 days with tempol (2 mM) or DL-buthionine-(S,R)-sulfoximine (BSO, 20 mM) in the drinking water. Using laser Doppler flowmetry, renal blood perfusion in the cortex and medulla (CBP, MBP) was measured during the infusion of Ap A. This induced a biphasic response in the CBP: a phase of rapid decrease was followed by one of rapid increase extended for 30 min in both the normocholesterolemic and hypercholesterolemic rats. The phase of decreased CBP was not affected by tempol or BSO in either group. Early and extended increases in CBP were prevented by tempol in the hypercholesterolemia rats, while, in the normocholesterolemic rats, only the extended increase in CBP was affected by tempol; BSO prevented extended increase in CBP in normocholesterolemic rats. MBP response is not affected by hypercholesterolemia. The hypercholesterolemic rats were characterized by increased urinary albumin and 8-isoPGF excretion. Moreover, BSO increased the urinary excretion of nephrin in the hypercholesterolemic rats but, similar to tempol, did not affect the excretion of albumin in their urine. The results suggest the important role of redox balance in the extracellular nucleotide regulation of the renal vasculature and glomerular injury in hypercholesterolemia.
Topics: Animals; Diet, High-Fat; Dinucleoside Phosphates; Hemodynamics; Hypercholesterolemia; Kidney; Lipids; Male; Oxidation-Reduction; Purinergic P2 Receptor Agonists; Rats; Rats, Wistar; Receptors, Purinergic P2; Renal Circulation
PubMed: 34110719
DOI: 10.14814/phy2.14888 -
Cell Reports Jun 2021Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of...
Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of mutations and transcription factor binding sites, we show that C/EBP binding sites are strongly enriched with [C > T]G mutations in cancer and adult stem cells, which is of special interest because C/EBPs regulate cell fate and differentiation. In vitro protein-DNA binding assay and structural modeling of the CEBPB-DNA complex show that the G·T mismatch in the core CG dinucleotide strongly enhances affinity of the binding site. We conclude that enhanced binding of C/EBPs shields CpG·TpG mismatches from DNA repair, leading to selective accumulation of [C > T]G mutations and consequent deterioration of the binding sites. This mechanism of targeted mutagenesis highlights the effect of a mutational process on certain regulatory sites and reveals the molecular basis of putative regulatory alterations in stem cells.
Topics: Adult Stem Cells; CCAAT-Enhancer-Binding Protein-alpha; Dinucleoside Phosphates; Humans; Mutation; Neoplasms
PubMed: 34107262
DOI: 10.1016/j.celrep.2021.109221 -
Acta Cardiologica Sep 2021Coronary artery bypass grafting (CABG) remains the gold standard treatment for mutivessel and left main coronary artery disease (CAD). Saphenous vein graft (SVG) patency...
BACKGROUND
Coronary artery bypass grafting (CABG) remains the gold standard treatment for mutivessel and left main coronary artery disease (CAD). Saphenous vein graft (SVG) patency is still a problem in CAD patients after CABG surgery. The Dual Antiplatelet Treatment (DAPT) score is a clinical prediction tool that predicts ischaemic and bleeding risk in CAD patients. The aim of this study is to investigate the relationship between DAPT score and SVG patency in CABG patients.
METHOD
This retrospective study enrolled a total of 398 patients (68 female; mean age 65.8 ± 9.1 years) with a history of CABG surgery. The study population was divided into two subgroups according to SVG patency. The DAPT score was calculated for each patients and compared between the two groups.
RESULTS
Coronary angiography revealed SVG disease in 212 patients and SVG patency in 186 patients. The rates of diabetes mellitus and hypertension, red cell distribution width values, DAPT Score, time interval after CABG and number of SVGs were significantly higher while LVEF was significantly lower in patients with SVG disease. The presence of diabetes mellitus, high DAPT score, long time interval after CABG and high number of SVGs were found to be independent predictors of SVG patency. DAPT score above 2.5 predicted SVG disease with a sensitivity of 77.1% and a specificity of 87.1% (AUC: 0.873; 95%CI: 0.823-0.924; < 0.001).
CONCLUSION
The DAPT score may provide useful information for SVG patency in CABG patients. Patients with high DAPT score should be followed up closely for SGV occlusion. DAPT score may be useful prior to CABG in determining the duration of dual anti-platelet therapy and in encouraging the use of arterial grafts with better patency.
Topics: Aged; Coronary Artery Bypass; Dinucleoside Phosphates; Female; Humans; Middle Aged; Retrospective Studies; Saphenous Vein; Vascular Patency
PubMed: 33880976
DOI: 10.1080/00015385.2021.1912248 -
PLoS Biology Apr 2021Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In...
Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG dinucleotide frequencies showed substantially enhanced replication compared to wild type. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG- or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wild-type virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on dinucleotide composition. However, the evolutionary pressure on the CpG dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arbo flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector.
Topics: A549 Cells; Aedes; Animals; Base Composition; Base Sequence; Cell Line; Chlorocebus aethiops; CpG Islands; Dinucleoside Phosphates; Evolution, Molecular; Genome, Viral; Host Adaptation; Host-Pathogen Interactions; Humans; Male; Mammals; Mice; Mice, Inbred C57BL; Mice, Knockout; Mosquito Vectors; RNA, Viral; Selection, Genetic; Vero Cells; Zika Virus; Zika Virus Infection
PubMed: 33872300
DOI: 10.1371/journal.pbio.3001201 -
Apoptosis : An International Journal on... Jun 2021Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of...
Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negative breast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1 cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.
Topics: Apoptosis; Cell Death; Cell Line, Tumor; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Membrane Proteins; Mitochondria; Protein Binding; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Triple Negative Breast Neoplasms
PubMed: 33840002
DOI: 10.1007/s10495-021-01669-x -
Frontiers in Cellular and Infection... 2021Tuberculosis (TB), caused by (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary...
Tuberculosis (TB), caused by (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary for preventing the prevalence of TB. In this study, a subunit vaccine of ESAT-6 formulated with c-di-AMP (ESAT-6:c-di-AMP) promoted mucosal and systemic immune responses in spleen and lung. ESAT-6:c-di-AMP inhibited the differentiations of CD8 T cells as well as macrophages, but promoted the differentiations of ILCs in lung. The co-stimulation also enhanced inflammatory cytokines production in MH-S cells. It was first revealed that ESAT-6 and c-di-AMP regulated autophagy of macrophages in different stages, which together resulted in the inhibition of Mtb growth in macrophages during early infection. After Mtb infection, the level of ESAT-6-specific immune responses induced by ESAT-6:c-di-AMP dropped sharply. Finally, inoculation of ESAT-6:c-di-AMP led to significant reduction of bacterial burdens in lungs and spleens of immunized mice. Our results demonstrated that subunit vaccine ESAT-6:c-di-AMP could elicit innate and adaptive immune responses which provided protection against Mtb challenge, and c-di-AMP as a mucosal adjuvant could enhance immunogenicity of antigen, especially for innate immunity, which might be used for new mucosal vaccine against TB.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; CD8-Positive T-Lymphocytes; Dinucleoside Phosphates; Immunity; Mice; Mycobacterium tuberculosis; Tuberculosis; Tuberculosis Vaccines; Vaccines, Subunit
PubMed: 33829000
DOI: 10.3389/fcimb.2021.647220 -
Beilstein Journal of Organic Chemistry 2021Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside β-cyanoethyl phosphite triester formed...
Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)--trimethylbutan-1-aminium iodide (N+ azide) or -toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.
PubMed: 33828619
DOI: 10.3762/bjoc.17.65 -
The Science of the Total Environment Aug 2021DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a...
DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a potential therapeutic target drug for many diseases, such as cancer, neurological, cardiovascular, and cerebrovascular diseases. However, the pharmacological action and specific mechanisms of DAPT are not clear. Planarians have strong regenerative capacity and can regenerate a new individual with a complete nervous system in one week. Thus, they are used as an ideal indicator of environmental toxicants and a novel model for studying neurodevelopmental toxicology. In this study, different concentrations and treatment times of DAPT are used to analyze the gene expression levels of major components in Notch pathway. The results show that the optimal concentration and exposure time of DAPT is 100 nM for 10 days in planarians and indicate that the inhibitory of DAPT treatment on Notch pathway is time- and concentration-dependent. The potential impact of DAPT is effectively analyzed by qPCR, WISH, and Immunofluorescence. The results indicate that DAPT exposure causes intact planarian wavy or swollen, and regenerative planarians asymmetric growth or muti-eye. Moreover, DAPT exposure increases cell proliferation and apoptosis, results in neurodevelopmental defects and dynamic changes of some marker genes. These results suggest that the balance of proliferation and apoptosis is disturbed, and then, affecting tissue homeostasis and differentiation. These findings demonstrate that DAPT has serious side effects in organisms and relies on Notch pathway to determine cell fate, it is cautious in the use of DAPT as a potential therapeutic approach for the disease in clinical trials.
Topics: Amyloid Precursor Protein Secretases; Animals; Dinucleoside Phosphates; Pharmaceutical Preparations; Planarians; Signal Transduction
PubMed: 33812110
DOI: 10.1016/j.scitotenv.2021.146735