Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways

In addition to being a very well-known ion pump, Na⁺, K⁺-ATPase is a cell–cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na⁺, K⁺-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.     

Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells

Background

Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO₂ nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO₂ Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results

Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO₂ NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion

The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-700) contains supplementary material, which is available to authorized users.     

Associations between Mode of HIV Testing and Consent,Confidentiality, and Referral: A Comparative Analysis in Four African Countries

Background

Recommendations about scaling up HIV testing and counseling highlight the need to provide key services and to protect clients'' rights, but it is unclear to what extent different modes of testing differ in this respect. This paper examines whether practices regarding consent, confidentiality, and referral vary depending on whether testing is provided through voluntary counseling and testing (VCT) or provider-initiated testing.

Methods and Findings

The MATCH (Multi-Country African Testing and Counseling for HIV) study was carried out in Burkina Faso, Kenya, Malawi, and Uganda. Surveys were conducted at selected facilities. We defined eight outcome measures related to pre- and post-test counseling, consent, confidentiality, satisfactory interactions with providers, and (for HIV-positive respondents) referral for care. These were compared across three types of facilities: integrated facilities, where testing is provided along with medical care; stand-alone VCT facilities; and prevention of mother-to-child transmission (PMTCT) facilities, where testing is part of PMTCT services. Tests of bivariate associations and modified Poisson regression were used to assess significance and estimate the unadjusted and adjusted associations between modes of testing and outcome measures. In total, 2,116 respondents tested in 2007 or later reported on their testing experience. High percentages of clients across countries and modes of testing reported receiving recommended services and being satisfied. In the unadjusted analyses, integrated testers were less likely to meet with a counselor before testing (83% compared with 95% of VCT testers; p<0.001), but those who had a pre-test meeting were more likely to have completed consent procedures (89% compared with 83% among VCT testers; p<0.001) and pre-test counseling (78% compared with 73% among VCT testers; p = 0.015). Both integrated and PMTCT testers were more likely to receive complete post-test counseling than were VCT testers (59% among both PMTCT and integrated testers compared with 36% among VCT testers; p<0.001). Adjusted analyses by country show few significant differences by mode of testing: only lower satisfaction among integrated testers in Burkina Faso and Uganda, and lower frequency of referral among PMTCT testers in Malawi. Adjusted analyses of pooled data across countries show a higher likelihood of pre-test meeting for those testing at VCT facilities (adjusted prevalence ratio: 1.22, 95% CI: 1.07–1.38) and higher satisfaction for stand-alone VCT facilities (adjusted prevalence ratio: 1.15; 95% CI: 1.06–1.25), compared to integrated testing, but no other associations were statistically significant.

Conclusions

Overall, in this study most respondents reported favorable outcomes for consent, confidentiality, and referral. Provider-initiated ways of delivering testing and counseling do not appear to be associated with less favorable outcomes for clients than traditional, client-initiated VCT, suggesting that testing can be scaled up through multiple modes without detriment to clients'' rights. Please see later in the article for the Editors'' Summary     

Identification of selective sweeps in closely related populations of the house mouse based on microsatellite scans

Genome scans of polymorphisms promise to provide insights into the patterns and frequencies of positive selection under natural conditions. The use of microsatellites as markers has the potential to focus on very recent events, since in contrast to SNPs, their high mutation rates should remove signatures of older events. We assess this concept here in a large-scale study. We have analyzed two population pairs of the house mouse, one pair of the subspecies Mus musculus domesticus and the other of M. m. musculus. A total of 915 microsatellite loci chosen to cover the whole genome were assessed in a prescreening procedure, followed by individual typing of candidate loci. Schlötterer's ratio statistics (lnRH) were applied to detect loci with significant deviations from patterns of neutral expectation. For eight loci from each population pair we have determined the size of the potential sweep window and applied a second statistical procedure (linked locus statistics). For the two population pairs, we find five and four significant sweep loci, respectively, with an average estimated window size of 120 kb. On the basis of the analysis of individual allele frequencies, it is possible to identify the most recent sweep, for which we estimate an onset of 400–600 years ago. Given the known population history for the French–German population pair, we infer that the average frequency of selective sweeps in these populations is higher than 1 in 100 generations across the whole genome. We discuss the implications for adaptation processes in natural populations.     

A rapid hypersensitive response associated with resistance in the willow Salix viminalis against the gall midge Dasineura marginemtorquens

Great genotypic variation in resistance against the gall midge Dasineura marginemtorquens Bremi (Diptera: Cecidomyiidae) exists within its willow host Salix viminalis L. (Salicaceae). In some resistant genotypes larvae die within 40 h after attempting to initiate galls. The present study tested the hypothesis that the hypersensitive response (HR) is involved in incompatible interactions between D. marginemtorquens and S. viminalis. By means of UV/blue light and visible light microscopy, we verified a rapidly (within 12 h after egg hatch) spreading cell death of an HR-type due to larval attack in resistant willow genotypes. Twelve h after egg hatch, the cell death had spread up to six cell layers in resistant S. viminalis genotypes whereas in susceptible genotypes only up to two cell layers were affected. In the groups of dead cells on the resistant genotypes accumulation of phenolics was observed within 24 h after egg hatch. The rapidity of the cell death induction, the early local accumulation of phenolic compounds, and the strong association of the cell death with larval mortality suggest that the formation of dead cells in resistant willow genotypes should be interpreted as a true HR.     

DPP4 Gene DNA Methylation in the Omentum is Associated With Its Gene Expression and Plasma Lipid Profile in Severe Obesity

Severely obese subjects with the metabolic syndrome (MS) have higher dipeptidyl peptidase‐4 (DPP4) expression in their visceral adipose tissue (VAT) compared to obese individuals without MS. We tested the hypothesis that methylation level of CpG sites in the DPP4 promoter CpG island in VAT was genotype‐dependent and associated with DPP4 mRNA abundance and MS‐related phenotypes. The VAT DNA was extracted in 92 severely obese premenopausal women undergoing biliopancreatic derivation for the treatment of obesity. Women were nondiabetic and none of them used medication to treat MS features. Cytosine methylation rates (%) of 102 CpG sites in the DPP4 CpG island were assessed by pyrosequencing of sodium bisulfite‐treated DNA. Methylation rates were >10% for CpG sites 94–102. Their mean methylation rate (%Meth_94–102) was different between genotypes for DPP4 polymorphisms rs13015258 (P = 0.001), rs17848915 (P = 0.0004), and c.1926 G>A (P = 0.001). The %Meth_94–102 correlated negatively with DPP4 mRNA abundance (r = ?0.25, P < 0.05) and positively with plasma high‐density lipoprotein (HDL) cholesterol concentrations (r = 0.22, P < 0.05), whereas DPP4 mRNA abundance correlated positively with plasma total‐/HDL‐cholesterol ratio (r = 0.25; P < 0.05). In the VAT of nondiabetic severely obese women, genotype‐dependent methylation levels of specific CpG sites in the DPP4 promoter CpG island were associated with DPP4 gene expression and variability in the plasma lipid profile. Higher DPP4 gene expression in VAT and its relationship with the plasma lipid profile may be explained by actually unknown DPP4 biological effect or, to another extent, may also be a marker of VAT inflammation known to be associated with metabolic disturbances.     

Changes in Sleeping and Basal Energy Expenditure and Substrate Oxidation Induced by Short Term Thyroxin Administration in Man

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n=7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180μg T₄/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T₄ dose (ΔEE: +8.5 ± 2.3%, Δ heart rate +16.1 ± 2.2%). The T₄ dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T₄ administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T₄ provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.     

Chloral hydrate effects on oogenesis in Marsilea vestita Hook. et Grev

Chloral hydrate [3.5 × 10^?2 M] applied to archegonium of Marsilea vestila during oogenesis modifies the asymmetry of the gametic mitoses and the nuclear migrations. Multinucleate female gametes are obtained whose polarity is disturbed. At the ultrastructural level, the distribution of the organelles is changed profoundly. The ‘fertilization cone’ disappears, and there is increased vacuolation associated with myelin figures in the cytoplasm and the appearance of filament bundles. The nuclei also contain vacuoles and myelin figures.