Viral Inhibition Mechanism Mediated by Surface-Modified Silica Nanoparticles

Vaccines and therapies are not available for several diseases caused by viruses, thus viral infections result in morbidity and mortality of millions of people every year. Nanoparticles are considered to be potentially effective in inhibiting viral infections. However, critical issues related to their use include their toxicity and their mechanisms of antiviral action, which are not yet completely elucidated. To tackle these problems, we synthesized silica nanoparticles with distinct surface properties and evaluated their biocompatibility and antiviral efficacy. We show that nanoparticles exhibited no significant toxicity to mammalian cells, while declines up to 50% in the viral transduction ability of two distinct recombinant viruses were observed. We designed experiments to address the mechanism of antiviral action of our nanoparticles and found that their hydrophobic/hydrophilic characters play a crucial role. Our results reveal that the use of functionalized silica particles is a promising approach for controlling viral infection and offer promising strategies for viral control

Adipose Tissue-Derived Stem Cells from Humans and Mice Differ in Proliferative Capacity and Genome Stability in Long-Term Cultures

Adipose tissue-derived stem cells (ASCs) are among the more attractive adult stem cell options for potential therapeutic applications. Here, we studied and compared the basic biological characteristics of ASCs isolated from humans (hASCs) and mice (mASCs) and maintained in identical culture conditions, which must be examined prior to considering further potential clinical applications. hASCs and mASCs were compared for immunophenotype, differentiation potential, cell growth characteristics, senescence, nuclear morphology, and DNA content. Although both strains of ASCs displayed a similar immunophenotype, the percentage of CD73(+) cells was markedly lower and CD31(+) was higher in mASC than in hASC cultures. The mean population doubling time was 98.08 +/- 6.15 h for hASCs and 52.58 +/- 3.74 h for mASCs. The frequency of nuclear aberrations was noticeably lower in hASCs than in mASCs regardless of the passage number. Moreover, as the cells went through several in vitro passages, mASCs showed changes in DNA content and cell cycle kinetics (frequency of hypodiploid, G0/G1, G2/M, and hyperdiploid cells), whereas all of these parameters remained constant in hASCs. Collectively, these results suggest that mASCs display higher proliferative capacity and are more unstable than hASCs in long-term cultures. These results underscore the need to consider specificities among model systems that may influence outcomes when designing potential human applications.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[01/0009-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05/54695-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05/57591-4]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/06784-4]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/57937-0]Ministerio da Ciencia e Tecnologia/Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/Ministerio da Saude/Departamento Ciencia e Tecnologia (MCT/CNPq/MS/DE-CIT)[552324/20005-1]Ministerio da Ciencia e Tecnologia/Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/Ministerio da Saude/Departamento Ciencia e Tecnologia (MCT/CNPq/MS/DE-CIT)[10120104096700

Opposing action of NCoR1 and PGC-1 alpha in mitochondrial redox homeostasis

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1 alpha and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1 alpha overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity

Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879’s mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor

Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: a developmental study

Submitted by Adagilson Silva ([email protected]) on 2017-06-26T13:18:14Z No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-06-26T14:23:58Z (GMT) No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5)Made available in DSpace on 2017-06-26T14:23:58Z (GMT). No. of bitstreams: 1 28231241 2017 xav-hyd.oa.pdf: 3679006 bytes, checksum: f24ae366311a286279f0f0d3a9c930eb (MD5) Previous issue date: 2017-02Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, BrasilThe teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes

ZIKV infects female organs, placentas and embryos from wild-type immunocompetent mice (FVB/NJ and C57BL/6J), despite the absence of detectable viremia.

<p>Maternal blood samples were collected at 1h (n = 5), 12h (n = 3) and 24h (n = 3) after intravascular ZIKV injection at 5.5 days <i>post-coitum</i> (dpc), or at 12.5 dpc. Further blood samples, maternal organs, placentas and embryos/fetuses were collected at the day of harvest 10.5 dpc (n = 4) and 11.5 dpc (n = 2), or collected at 16.5 dpc (n = 4) in females injected at 12.5 dpc. Samples were assessed for viral load using a tissue culture infectious dose (TCID₅₀) assay in Vero cells. Embryos and respective placentas (n = 8) and maternal organ samples from two 16.5 dpc ZIKV-injected pregnant females (injected in 12.5 dpc), as well as two 12.5 PBS-injected control females were assessed for viral content using a TCID₅₀ assay. ZIKV was detected in the spleen of both females and in the liver of one female. Notably, all placentas had significant amounts of ZIKV, whereas just half of the embryos were ZIKV positive. No malformations were observed.</p

Morphometric analyses in three litters of ZIKV-injected pregnant mouse females.

<p>(<b>A-C</b>) To better normalize the morphometric results we plotted Crown rump length (CRL) and Occipital-frontal diameter (OFD) as a function of individual embryonic/fetal stages, rather than as nominal litter stages. All conceptuses were classified according to Kauffman [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.ref020" target="_blank">20</a>] and Theiler [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.ref022" target="_blank">22</a>]. (<b>A</b>) CRL data. (<b>B</b>) OFD data. (<b>C</b>) OFD/CRL data. All data are displayed in relation to intervals defined as the average ± three standard deviations of PBS-injected (light grey), or reference controls (dark grey). Stage normalization eliminated all evidence for specific changes in cephalic proportions, but for two conceptuses. One fetus (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.g007" target="_blank">Fig 7H</a>) displayed an abnormally increased OFD/CRL, which resulted from cephalic collapse in the cranio-caudal axis (arrowhead), while the other fetus (shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005363#pntd.0005363.g006" target="_blank">Fig 6D</a>) constitutes the only specific evidence for microcephaly in our study (arrow).</p

Fetuses from FVB/NJ pregnant female injected with ZIKV at 9.5 days <i>post coitum</i> (dpc).

<p>Fetuses were harvested at 16.5 dpc. (<b>A-H</b>) Fetuses are displayed in an order of increased severity of developmental malformations. (<b>I-M</b>) Outwardly normal littermates. (<b>N-P</b>) Fetal phenotypes recorded immediately after harvesting and initial dissection. Prominent features of malformed embryos ranged from vascular rarefaction and pallor in the cephalic region (<b>A</b>, <b>P</b>) (square bracket), generalized edema (<b>B</b>, <b>C</b>) (arrows) to complete disruption of normal development (<b>E</b>-<b>H</b>). Note the intra amniotic hemorrhage in <b>N</b>, clouding of the amniotic fluid in <b>O</b> and the preferential pallor and/or vascular rarefaction in the cephalic region in <b>P</b> (square bracket). White arrowhead indicates abnormal forelimb postures suggestive of arthrogryposis. Scale bar, 2.0 mm.</p