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The insertion of protein domains into membranes is an important step in many membrane remodeling processes, for example in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. Total area changes in lipid monolayers can be measured on a Langmuir film balance. Finding the area per inserted protein however proves challenging for two reasons: The number of inserted proteins must be determined without disturbing the binding equilibrium and the change in the film area can be very small. Here we address both issues using Fluorescence Correlation Spectroscopy (FCS): Firstly, by labeling a fraction of the protein molecules fluorescently and performing FCS experiments directly on the monolayer, the number of inserted proteins is determinedin situwithout having to rely on invasive techniques, such as collecting the monolayer by aspiration. Secondly, by using another FCS color channel and adding a small fraction of fluorescent lipids, the reduction in fluorescent lipid density accompanying protein insertion can be monitored to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation, in a lipid monolayer. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2was obtained for Sar1 in monolayers from a lipid mixture typically used in reconstitution, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to be applied also to laterally heterogeneous monolayers and bilayers.Statement of SignificanceWe show that two color Fluorescence Correlation Spectroscopy (FCS) measurements can be applied to the binding of a protein to a lipid monolayer on a Langmuir film balance in order to determine the protein insertion area. One labelling color was used to determine the number of bound proteins and the other one to monitor the area expansion of the lipid monolayer upon protein binding. A strategy for the FCS data analysis is provided, which includes focal area calibration by raster image correlation spectroscopy and a framework for applying z-scan FCS and including free protein in the aqueous subphase. This approach allows determining an area occupied by a protein in a quasi-planar model membrane from a local, non-invasive, optical measurement.

Today, we witness the progress toward global COVID-19 vaccinations organized by countries worldwide. Experts say a mass vaccination plan is the only effective antidote against the spread of SARS-COV-2. However, a part of the world population refuses vaccination. The present study aimed to understand the impact of some individual variables on the intention to get vaccinated. Through a serial mediation model, we tested the influence of fear of COVID-19 on the intention to get vaccinated and the serial mediating effect of existential anxiety and conspiracy beliefs. Via a cross-sectional design this research was conducted with the participation of 223 French adults (Female: 69.5%; Male: 30.5%; Mage = 30.26, SD = 13.24; range: 18–75 years) who responded to an online survey. The results showed a positive relationship between fear of COVID-19 and intention to get vaccinated; however, when this fear was associated with high levels of existential anxiety through conspiracy beliefs, the intention to get vaccinated decreased. Our findings were in line with Terror Management Health Model, which states that, in facing health threats, humans may strive to reduce their own perceived vulnerability not only by engaging in healthy behaviors but also denying or avoiding death anxiety, as anti-vaxxers do.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation. The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown. In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host. We found that InvD is induced at 37 °C and expressed in vivo 2–4 days after infection, indicating that InvD is a virulence factor. During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues. The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure. We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the adhesion domain. Phage display panning and flow cytometry data further revealed that InvD exhibits a preferential binding specificity toward antibodies with VH3/VK1 variable domains and that it is specifically recruited to a subset of B cells. This finding suggests that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors. In summary, our results provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host.

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3h exposure at 39°C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca2+ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMALl-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork. (C) 2015 Elsevier B.V. All rights reserved.

“Appropriation” has been studied as a mechanism by which space is transformed into place. The present research tested two models of appropriation and place attachment among university students (n = 247), one in their university's neighborhood and the other in Paris as a whole. In both models, the relation between time spent in the setting and attachment to the setting was mediated by students' appropriating behaviors (the mean of three measures: exploration through route variety, exploration via diversity of destinations, and knowledge of landmarks). Results were complicated, with both models revealing combinations of full and partial mediation but both indicating that time alone was not related to attachment. Instead, appropriating behaviors were related to higher attachment. Both models support the idea that appropriation is a mechanism by which attachment develops, thereby encouraging additional research on this important and complex process.

International audience; Adult attachment style has only recently been considered as having a role in explaining work behavior. The present research aimed to explore the impact of adult attachment style, assessed by the Adult Attachment Interview (AAI), on organizational commitment (OC) and on adult attachment in the workplace (AAW). We hypothesized that a secure attachment style would be positively related to affective and normative commitment, while preoccupied and avoidant styles would be negatively related to affective commitment; we also hypothesized that there would be a correspondence between the AAI categories and the AAW dimensions. Using the AAI categories as group variable, analysis of average OC and AAW scores confirmed the hypotheses. Secure workers had a higher mean score for affective commitment than avoidant and preoccupied workers; normative commitment was higher in avoidant than in secure and preoccupied workers; continuance commitment was higher in preoccupied than in secure and avoidant workers. Moreover, AAI categories converged with AAW dimensions: secure workers had higher secure AAW scores than avoidant and preoccupied workers; avoidant workers had higher avoidant AAW scores than secure and preoccupied workers; preoccupied workers had higher preoccupied AAW scores than secure and avoidant workers.

C8, a short peptide characterized by three regularly spaced Trp residues, belongs to the membrane-proximal external functional domains of the feline immunodeficiency virus coat protein gp36. It elicits antiviral activity as a result of blocking cell entry and exhibits membranotropic and fusogenic activities. Membrane-proximal external functional domains of virus coat proteins are potential targets in the development of new anti-HIV drugs that overcome the limitations of the current anti-retroviral therapy. In the present work, we studied the conformation of C8 and its interaction with micellar surfaces using circular dichroism, nuclear magnetic resonance and fluorescence spectroscopy. The experimental data were integrated by molecular dynamics simulations in a micelle–water system. Our data provide insight into the environmental conditions related to the presence of the fusogenic peptide C8 on zwitterionic or negatively charged membranes. The membrane charge modulates the conformational features of C8. A zwitterionic membrane surface induces C8 to assume canonical secondary structures, with hydrophobic interactions between the Trp residues and the phospholipid chains of the micelles. A negatively charged membrane surface favors disordered C8 conformations and unspecific superficial interactions, resulting in membrane destabilization.

Regulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In a previous study we showed that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as here documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork.

Hepatitis C virus (HCV) infection induces a state of oxidative stress more pronounced than that observed in many other inflammatory diseases. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen species and a progressive metabolic adaptive response. Evidence is provided for a positive feed-back mechanism between alterations of calcium and redox homeostasis. This likely involves deregulation of the mitochondrial permeability transition and induces progressive dysfunction of cellular bioenergetics. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumor promoters on preexisting "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown. In this study we investigated the effect of Hg(II) (HgCl(2)) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl(2) as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl(2)-treated keratinocytes exhibited a decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species, albeit no effect on the respiratory chain activity was observed. Treatment of HgCl(2)-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS overproduction and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP. Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signaling in HgCl(2)-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.

Functional biochemical tests are the gold standard for the diagnosis of mitochondria-related diseases. However, the availability of the biological samples from patients' tissues represents a severe limitation to the number of screenable enzymatic activities. In this study we developed a fluorescent probe-assisted microscopy protocol enabling to assess the ΔΨ(m)-generating capacity by mitochondria immobilized on a glass surface at the single organelle resolution-level. The advantage of this assay over others is to scale-down the amount of the biological sample required to test in a short time the functional activity of all the components of the oxidative phosphorylation system without loss of accuracy. Furthermore, the distribution of a given enzymatic activity can also be evaluated within the mitochondrial population enabling to measure the level of functional heterogeneity of the respiratory chain dysfunction.

This study aimed to validate, in situ, proposed mechanisms of bupivacaine cytotoxicity pointing to impairment of the mitochondrial oxidative metabolism. High resolution oxymetry, carried out on a panel of cell cultures, revealed a dual dose- and time-dependent effect of bupivacaine consisting of uncoupling of the mt Delta mu(H+)-controlled respiratory rates in a cyclosporine A-insensitive manner and further inhibition of the respiratory rates. Intriguingly, a relatively small decrease on the mt Delta Psi (about 20 mV) was sufficient to account for both the bupivacaine- and the FCCP-mediated impairment of the oxidative phosphorylation coupling thereby supporting a common protonophoric mechanism of action. The bupivacaine-induced depression of the cell respiration related to specific inhibition of complexes I and III and accompanied with production of reactive oxygen species. Importantly, inhibition of the respiratory chain complexes was prevented by antioxidant treatment and reversed following removal of the anaesthetic thereby suggesting an oxidant-mediated feed-back mechanism reinforcing the primary inhibitory action of the anaesthetic.

Introduction Hepatocellular carcinoma (HCC) represents the sixth leading cancer and the third most common cause of death from cancer. Many different aetiological factors are involved in the development of HCC, which may be modulated by both estrogens and androgens hormones during its initiation, progression and metastasis. The misuse of anabolic androgenic steroids (AAS) is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas, and is considered a factor risk of developing hepatic sex hormone related tumours. The purpose of this study was to investigate the role of Nandrolone, one of the most commonly used AAS, in regulating proliferation and differentiation of HCC. Material and methods Human HCC cell line HepG2 was treated with Nandrolone, a synthetic androgen ligand, for 48 hs and its viability and proliferation was assessed by MTS and cell cycle analysis, respectively. The expression of protein involved in cell cycle regulation and differentiation markers were analysed by western blot and real time PCR. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed using Seahorse XF96 extracellular flux analyzer. Respiratory chain complex activities were assayed spectrophotometrically. Stemness surface markers expression was detected by FACSCalibur flow cytometer. Results and discussions Nandrolone treatment caused cell growth inhibition associated to a downregulation of cyclin D1 and an upregulation of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 leading to cell cycle arrest in the G2 phase. Moreover, a significant overall impairment of mitochondrial functions, resulting in a reduced OCR and impairment of OXPHOS complexes activities were also observed, thus suggesting a role in the control of the metabolic reprogramming. Finally, a significant increase of the stemness markers was detected following Nandrolone treatment, also confirmed in additional human stem cell types and in an in vivo mouse model. Conclusion Nandrolone shows a strong anti-proliferative effect in differentiated tumour cells, promoting cancer cells stemness through cellular metabolic reprogramming. These results could have important public health implications in order to improve the primary prevention such as revising altered lifestyles, like AAS abuse.