scholarly journals Parvovirus B19 Uncoating Occurs in the Cytoplasm without Capsid Disassembly and It Is Facilitated by Depletion of Capsid-Associated Divalent Cations

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 430 ◽  
Author(s):  
Oliver Caliaro ◽  
Andrea Marti ◽  
Nico Ruprecht ◽  
Remo Leisi ◽  
Suriyasri Subramanian ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Parvovirus B19 ◽  
Significant Proportion ◽  
Endosomal Escape ◽  
Short Exposure ◽  
Nuclear Fraction ◽  
Nuclear Entry ◽  
Thaw Cycle ◽  
Capsid Disassembly

Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

Estradiol: Specific Binding by Pituitary Nuclear Fraction in vitro

Science ◽  
1969 ◽  
Vol 165 (3892) ◽  
pp. 496-498 ◽  
Author(s):  
W. W. Leavitt ◽  
J. P. Friend ◽  
J. A. Robinson
Keyword(s):  
Specific Binding ◽  
Nuclear Fraction

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

scholarly journals Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 382
Author(s):  
Amisha Sanghani ◽  
Konstantinos N. Kafetzis ◽  
Yusuke Sato ◽  
Salsabil Elboraie ◽  
Julia Fajardo-Sanchez ◽  
...  
Keyword(s):  
Encapsulation Efficiency ◽  
Serum Response Factor ◽  
Cationic Lipid ◽  
Endosomal Escape ◽  
The Novel ◽  
Small Interfering Rnas ◽  
Glaucoma Filtration Surgery ◽  
Promising Therapeutic Approach ◽  
Cleavable Peptide

The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.

scholarly journals CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

2021 ◽  
Vol 22 (2) ◽  
pp. 912
Author(s):  
Nabila Seddiki ◽  
John Zaunders ◽  
Chan Phetsouphanh ◽  
Vedran Brezar ◽  
Yin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Proportion ◽  
Long Term Memory ◽  
Ki 67 ◽  
Memory Cd4 T Cells ◽  
Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19

2013 ◽  
Vol 193 (1) ◽  
pp. 215-225 ◽  
Author(s):  
Raphael Wolfisberg ◽  
Nico Ruprecht ◽  
Christoph Kempf ◽  
Carlos Ros
Keyword(s):  
In Vitro Propagation ◽  
Parvovirus B19 ◽  
Human Parvovirus B19 ◽  
Genome Encapsidation

High Rate of Negative Results of Tuberculin and QuantiFERON Tests Among Individuals With a History of Positive Skin Test Results

2006 ◽  
Vol 27 (5) ◽  
pp. 436-441 ◽  
Author(s):  
Lloyd N. Friedman ◽  
Esther R. Nash ◽  
June Bryant ◽  
Susan Henry ◽  
Julia Shi ◽  
...  
Keyword(s):  
Skin Test ◽  
Significant Proportion ◽  
High Rate ◽  
Negative Group ◽  
Tuberculosis Screening ◽  
Positive Skin ◽  
Screening Programs ◽  
History Of ◽  
Positive Results

Objectives.To evaluate individuals at high risk for tuberculosis exposure who had a history of a positive tuberculin skin test (TST) result in order to determine the prevalence of unsuspected negative TST results. To confirm these findings with the QuantiFERON-TB test (QFT), an in vitro whole-blood assay that measures tuberculin-induced secretion of interferon-γ.Methods.This survey was conducted from November 2001 through December 2003 at 3 sites where TST screening is regularly done. Detailed histories and reviews of medical records were performed. TSTs were placed and read by 2 experienced healthcare workers, and blood was drawn for QFT. Any subject with a negative result of an initial TST during the study (induration diameter, <10 mm) underwent a second TST and a second QFT. The TST-negative group comprised individuals for whom both TSTs had an induration diameter of <10 mm. The confirmed-negative group comprised individuals for whom both TSTs yielded no detectable induration and results of both QFTs were negative.Results.A total of 67 immunocompetent subjects with positive results of a previous TST were enrolled in the study. Of 56 subjects who completed the TST protocol, 25 (44.6%; 95% confidence interval [CI], 31.6%-57.6%) were TST negative (P<.001). Of 31 subjects who completed the TST protocol and the QFT protocol, 8 (25.8%; 95% CI, 10.4%-41.2%) were confirmed negative (P<.005).Conclusions.A significant proportion of subjects with positive results of a previous TST were TST negative in this study, and a subset of these were confirmed negative. These individuals' TST status may have reverted or may never have been positive. It will be important in future studies to determine whether such individuals lack immunity to tuberculosis and whether they should be considered for reentry into tuberculosis screening programs.

scholarly journals All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2807-2819 ◽  
Author(s):  
Friederike Klemke ◽  
Gabriele Beyer ◽  
Linda Sawade ◽  
Ali Saitov ◽  
Thomas Korte ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Enzymic Activity ◽  
Phosphoryl Group ◽  
Nitrogen Deprivation ◽  
Ping Pong ◽  
Nucleoside Triphosphates ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Promoter Studies

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with k cat = 48.2 s−1 at pH 7.5 and 28 °C and K M = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

scholarly journals The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 606
Author(s):  
Ieva Rinkūnaitė ◽  
Egidijus Šimoliūnas ◽  
Daiva Bironaitė ◽  
Rasa Rutkienė ◽  
Virginija Bukelskienė ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Parvovirus B19 ◽  
Acute Infection ◽  
Endothelial Damage ◽  
Precursor Cells ◽  
Unique Region ◽  
Erythroid Precursor ◽  
Active Replication

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats’ pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

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