5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the ...scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.
The knowledge of soil-geosynthetic interface behaviour is a key point in the design of geosynthetic-reinforced soil structures. The pullout ultimate limit state can be reproduced conveniently by ...means of pullout tests performed with large-size laboratory apparatuses, which allow studying the interaction mechanisms that develop in the anchorage zone. During the service life of geosynthetic-reinforced soil structures, reinforcements may be subjected to long-term cyclic vehicular loads or short-term seismic loads in addition to dead loadings, such as the structure's self-weight and other sustained loads. In order to study the influence of a cyclic loading history (a sinusoidal function with fixed amplitude A, number of cycles N and frequency f) on the post-cyclic peak pullout resistance, the writers carried out a series of multi-stage pullout tests on a high density polyethylene extruded uniaxial geogrid embedded in a compacted granular soil for different vertical effective stress σ′v values. Moreover, the stability of the soil-geosynthetic interface from a point of view linked to the cyclic loading application has also been investigated. Test results showed that the design pullout resistance parameters are affected by the applied cyclic loading history for specific combined conditions (A, N and σ′v) and it should be taken into account for designing geosynthetic reinforced soil structures.
At present, the design of geosynthetic-reinforced soil structures is executed with reference to the tensile strength of the reinforcement obtained from in-air short-term tensile tests, decreasing ...this value by means of several factors. Among these, the creep effect resulting from in-air tensile creep tests reduces tensile strength the most. Consequently, this procedure does not take into account the effects of soil confinement and interaction on the tensile response of the reinforcements. This paper illustrates a new large-scale pullout prototype apparatus, with the capacity to investigate the behaviour of a geosynthetic reinforcement embedded in a compacted soil and subject to a tensile load kept constant over time. The apparatus allows the verification of how the soil can modify the prediction of the long-term behaviour of geosynthetics. Results in terms of confined tensile strains were analysed, and the comparison of those values with the strains obtained by in-air tensile creep tests has led to the conclusion that the creep reduction factor might be conservative. Moreover, the confined tensile strains were related to the apparent coefficients of friction to propose a new procedure capable of determining the design interaction parameter under long-term pullout load as a function of the allowable reinforcement strains.
The most common cancers, including breast and skin, disseminate initially through the lymphatic system, yet the mechanisms by which tumor cells home towards, enter and interact with the lymphatic ...endothelium remain poorly understood. Transmural and luminal flows are important biophysical cues of the lymphatic microenvironment that can affect adhesion molecules, growth factors and chemokine expression as well as matrix remodeling, among others. Although microfluidic models are suitable for in vitro reconstruction of highly complex biological systems, the difficult assembly and operation of these systems often only allows a limited throughput. Here we present and characterize a novel flow chamber which recapitulates the lymphatic capillary microenvironment by coupling a standard Boyden chamber setup with a micro-channel and a controlled fluidic environment. The inclusion of luminal and transmural flow renders the model more biologically relevant, combining standard 3D culture techniques with advanced control of mechanical forces that are naturally present within the lymphatic microenvironment. The system can be monitored in real-time, allowing continuous quantification of different parameters of interest, such as cell intravasation and detachment from the endothelium, under varied biomechanical conditions. Moreover, the easy setup permits a medium-high throughput, thereby enabling downstream quantitative analyses. Using this model, we examined the kinetics of tumor cell (MDA-MB-231) invasion and transmigration dynamics across lymphatic endothelium under varying flow conditions. We found that luminal flow indirectly upregulates tumor cell transmigration rate via its effect on lymphatic endothelial cells. Moreover, we showed that the addition of transmural flow further increases intravasation, suggesting that distinct flow-mediated mechanisms regulate tumor cell invasion.
Folic acid is a nutrient essential for embryonic development. Folate deficiency can cause embryonic lethality or neural tube defects and orofacial anomalies. Folate receptor 1 (Folr1) is a folate ...binding protein that facilitates the cellular uptake of dietary folate. To better understand the biological processes affected by folate deficiency, gene expression profiles of gestational day 9.5 (gd9.5) Folr1−/− embryos were compared to those of gd9.5 Folr1+/+ embryos. The expression of 837 genes/ESTs was found to be differentially altered in Folr1−/− embryos, relative to those observed in wild-type embryos. The 837 differentially expressed genes were subjected to Ingenuity Pathway Analysis. Among the major biological functions affected in Folr1−/− mice were those related to ‘digestive system development/function’, ‘cardiovascular system development/function’, ‘tissue development’, ‘cellular development’, and ‘cell growth and differentiation’, while the major canonical pathways affected were those associated with blood coagulation, embryonic stem cell transcription and cardiomyocyte differentiation (via BMP receptors). Cellular proliferation, apoptosis and migration were all significantly affected in the Folr1−/− embryos. Cranial neural crest cells (NCCs) and neural tube explants, grown under folate-deficient conditions, exhibited marked reduction in directed migration that can be attributed, in part, to an altered cytoskeleton caused by perturbations in F-actin formation and/or assembly. The present study revealed that several developmentally relevant biological processes were compromised in Folr1−/− embryos.
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•Gene expression profiling was determined in Folate receptor 1 KO (Folr1−/−) embryos.•837 genes were differentially expressed in Folr1−/−, relative to Folr1+/+, embryos.•Increased apoptosis and decreased proliferation were noted in Folr1−/− cranial NCCs.•Directed migration of cranial NCCs was markedly affected in Folr1−/− embryos.•F-actin mediated cytoskeletal changes may impair NCC migration in Folr1−/− embryos.
Objective
Normal development of the embryonic orofacial region requires precise spatiotemporal coordination between numerous genes. MicroRNAs represent small, single-stranded, non-coding molecules ...that regulate gene expression. This study examines the role of microRNA-22 (miR-22) in murine orofacial ontogeny.
Methods
Spatiotemporal and differential expression of miR-22 (mmu-miR-22-3p) within the developing secondary palate was determined by in situ hybridization and quantitative real-time PCR, respectively. Bioinformatic approaches were used to predict potential mRNA targets of miR-22 and analyze their association with cellular functions indispensable for normal orofacial ontogeny. An in vitro palate organ culture system was used to assess the role of miR-22 in secondary palate development.
Results
There was a progressive increase in miR-22 expression from GD12.5 to GD14.5 in palatal processes. On GD12.5 and GD13.5, miR-22 was expressed in the future oral, nasal, and medial edge epithelia. On GD14.5, miR-22 expression was observed in the residual midline epithelial seam (MES), the nasal epithelium and the mesenchyme, but not in the oral epithelium. Inhibition of miR-22 activity in palate organ cultures resulted in failure of MES removal. Bioinformatic analyses revealed potential mRNA targets of miR-22 that may play significant roles in regulating apoptosis, migration, and/or convergence/extrusion, developmental processes that modulate MES removal during palatogenesis.
Conclusions
Results from the current study suggest a key role for miR-22 in the removal of the MES during palatogenesis and that miR-22 may represent a potential contributor to the etiology of cleft palate.
Environmental and genetic factors contribute significantly to the etiology of orofacial clefting, which is one of the most common of human congenital craniofacial malformations. Current biological ...thought now recognizes that epigenetics represents a fundamental contributing process in embryogenesis. Indeed, many of the mechanisms whereby environmental insults affect key pathways crucial for proper embryonic growth and development are increasingly thought to be mediated via the epigenome. Epigenetic regulators, such as microRNAs (miRNAs), play vital roles in the ontogeny of the orofacial region. Evidence for this comes from conditional knockouts of Dicer or DGCR8, genes encoding key enzymes in the miRNA biosynthetic machinery, in neural crest cells. Such knockouts result in a range of craniofacial/orofacial anomalies, including cleft palate and cleft lip. Epigenetic pathways may thus represent key vehicles in the regulation, and misregulation, of gene expression during normal and abnormal orofacial embryogenesis. Significant strides have been made in the last decade in identifying miRNAs and their target genes involved in lip and palate morphogenesis. Such morphogenetic processes include apoptosis, cell proliferation, cell differentiation, and epithelial-mesenchymal transition (EMT). While some of the miRNA-target gene interactions have been functionally validated, many exhibit causal relationships that await functional confirmation. A plethora of genes associated with cleft palate/cleft lip have now been identified that provides a veritable treasure trove of information that could be harnessed to identify novel miRNA candidates for further analysis. In this review, we summarize studies identifying miRNAs involved in various aspects of lip and palate morphogenesis and whose aberrant expression may result in orofacial clefts.
Water resources contaminated with wastewater are an important source for the dissemination of enteric viruses with an impact on the health of the population. The aim of the study was to assess the ...viral contamination of freshwater from a dam in Argentina by using infectious enterovirus detection, viral RNA amplification, and a genetic characterization of five enteric viruses associated with diarrhea and hepatitis. Enterovirus infectivity (iEV) was evaluated by cell culture and direct immunofluorescence. The detection of the viral genome of rotavirus (RV), human astrovirus (HAstV), norovirus (NoV), hepatitis A virus (HAV), and hepatitis E virus (HEV) was performed by reverse transcriptase PCR (RT-PCR). A total of 48 water samples from 4 monitoring points on the body of the dam from January to December 2012 and 66 water samples from 3 tourist beaches on the edge of the dam from October 2013 to October 2015 were collected monthly. During the first period, the overall viral frequency detection was 52.1% for group A RV, 50% for HAstV, 60.4% for NoV, 22.9% for HAV, 2.1% for HEV, and 64.6% for iEV. The overall frequency detection for the second sampling was 18.2% for RV and HAstV, 31.8% for NoV, 7.57% for HEV, and 66.7% for iEV. There was no detection of HAV during this period. The genotypes and genogroups detected through the study correlated with the most common genomic variants associated with human gastrointestinal and hepatitis illnesses. The results obtained could alert the health systems and environmental sanitation to make decisions for viral control and prevention in our environment.
The study shows the impact of anthropic contamination of one of the most important tourist water resources in Argentina. This course of recreational water would be a favorable scenario for infection, as well as a reservoir for the enteric viruses, creating a risk for the population exposed to these waters. The results obtained could alert the health systems and environmental sanitation to make decisions for the control and prevention of viral diseases in this environment.
Hyperthermic intraperitoneal chemotherapy (HIPEC) is advised as a treatment option for epithelial ovarian cancer (EOC) with peritoneal carcinomatosis. This study was designed to define the ...pharmacokinetics of cisplatin (CDDP) and paclitaxel (PTX) administered together during HIPEC.
Thirteen women with EOC underwent cytoreductive surgery (CRS) and HIPEC, with CDDP and PTX. Blood, peritoneal perfusate and tissue samples were harvested to determine drug exposure by high-performance liquid chromatography and matrix-assisted laser desorption ionization imaging mass spectrometry (IMS).
The mean maximum concentrations of CDDP and PTX in perfusate were, respectively, 24.8±10.4 μg ml(-1) and 69.8±14.3 μg ml(-1); in plasma were 1.87±0.4 μg ml(-1) and 0.055±0.009 μg ml(-1). The mean concentrations of CDDP and PTX in peritoneum at the end of HIPEC were 23.3±8.0 μg g(-1) and 30.1±18.3 μg(-1)g(-1), respectively. The penetration of PTX into the peritoneal wall, determined by IMS, was about 0.5 mm. Grade 3-4 surgical complications were recorded in four patients, five patients presented grade 3 and two patients presented grade 4 hematological complications.
HIPEC with CDDP and PTX after CRS is feasible with acceptable morbidity and has a favorable pharmacokinetic profile: high drug concentrations are achieved in peritoneal tissue with low systemic exposure. Larger studies are needed to demonstrate its efficacy in patients with microscopic postsurgical residual tumours in the peritoneal cavity.
•NDGA has in vitro virucidal and antiviral activity against DENV1.•The NDGA antiviral effect seems to be intracellular and during the first two hours post DENV internalization.•NDGA affects the ...amount of intracellular RNA of DENV1, and a decrease in the labeling of E DENV1 protein with NDGA treatment was also observed.
Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4.
We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity.
The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes.
NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process.
The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.
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