Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~ 23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (− 36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA + polyunsaturated FA):(saturated FA + TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.     

β‐amyloid context intensifies vascular smooth muscle cells induced inflammatory response and de‐differentiation

Several studies have shown that the accumulation of β‐amyloid peptides in the brain parenchyma or vessel wall generates an inflammatory environment. Some even suggest that there is a cause‐and‐effect relationship between inflammation and the development of Alzheimer's disease and/or cerebral amyloid angiopathy (CAA). Here, we studied the ability of wild‐type Aβ_1‐40‐peptide (the main amyloid peptide that accumulates in the vessel wall in sporadic forms of CAA) to modulate the phenotypic transition of vascular smooth muscle cells (VSMCs) toward an inflammatory/de‐differentiated state. We found that Aβ_1‐40‐peptide alone neither induces an inflammatory response, nor decreases the expression of contractile markers; however, the inflammatory response of VSMCs exposed to Aβ_1‐40‐peptide prior to the addition of the pro‐inflammatory cytokine IL‐1β is greatly intensified compared with IL‐1β‐treated VSMCs previously un‐exposed to Aβ_1‐40‐peptide. Similar conclusions could be drawn when tracking the decline of contractile markers. Furthermore, we found that the mechanism of this potentiation highly depends on an Aβ_1‐40 preactivation of the PI₃Kinase and possibly NFκB pathway; indeed, blocking the activation of these pathways during Aβ_1‐40‐peptide treatment completely suppressed the observed potentiation. Finally, strengthening the possible in vivo relevance of our findings, we evidenced that endothelial cells exposed to Aβ_1‐40‐peptide generate an inflammatory context and have similar effects than the ones described with IL‐1β. These results reinforce the idea that intraparietal amyloid deposits triggering adhesion molecules in endothelial cells, contribute to the transition of VSMCs to an inflammatory/de‐differentiated phenotype. Therefore, we suggest that acute inflammatory episodes may increase vascular alterations and contribute to the ontogenesis of CAA.     

Pediatric dental emergency management and parental treatment preferences during COVID-19 pandemic as compared to 2019

Pediatric dental emergency management were temporarily suspended during the COVID-19 pandemic, which worsened urgent dental needs. This retrospective study investigated the management of pediatric emergencies during COVID-19 lockdown and the trends in parental preferences from March to July in 2019 and 2020. Pediatric dental emergencies managed during pandemic was collated, procedures were categorized (emergency, restorative, preventive, elective) and trends in parental treatment preference was compared from March-July 2019/2020. Bivariate analysis was performed using fisher-exact test and statistical significance was set at 5%. Total 1081 children were treated during COVID-19 lockdown, and 1509 procedures were performed, of which 20.8% were emergency, 42% restorative, 24.4% preventive, 12.6% elective. In 2019, 7462 children were treated; and except for emergency (10.6%), other procedures were comparable to 2020. Extractions (267) predominated in 2020 followed by sealants (195); but in 2019, pulectomy (1268), scaling (1251) were predominant. None of the residents who performed aerosol procedures got infected with COVID-19 during the lockdown. Emergency dental needs among pediatric patients were very high during the COVID-19 pandemic in South India, and there was not much change in the trend in parental treatment preference in 2019 and 2020. Further, aerosol procedures did not increase the risk of COVID-19 during the pandemic provided proper universal precautions were followed.     

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

Obesity, insulin resistance and diabetes in the sand rat exposed to a hypercaloric diet; possible protective effect for IL1-β

It is well established that, upon changing their natural desert low caloric (succulent halophilic plants) to a regular laboratory high caloric diet, sand rats undergo various phenotypic changes depending on their genetic background and including obesity and various degrees of insulin resistance. Our aim was to investigate the acute effects of Interleukin-1β (IL-1β) and Interferon-γ (IFN-γ) on glucose-induced insulin secretion in normal lean sand rats maintained on their natural diet and in obese insulin resistant normoglycemic or type 2 diabetic animals after a 9-month high caloric diet. Animals were fed either a low or a high caloric diet; after 9 months, pancreatic islets were isolated and incubated in the presence of increasing cytokine concentrations. At the end of the high-energy diet, animals were all over-weight, and probably due to a different genetic background, they displayed either insulin resistance, hyperinsulinemia and normoglycemia or a marked type-2 diabetic state. Pancreatic islets from obese insulin resistant normoglycemic animals were much more sensitive and responsive to IL-1β when compared to lean controls. The cytokine was inefficient in diabetic islets. In conclusion, the markedly increased insulinotropic effect of IL-1β in obese diabetes-resistant sand rat could participate and be involved in pancreatic β-cell hyperactivity that compensates for insulin resistance and thereby prevent the development of type 2 diabetes in these animals.     

Loss of homologous recombination or non-homologous end-joining leads to radial formation following DNA interstrand crosslink damage

High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways.     

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.     

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.     

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.