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Viruses must overcome the interferon-mediated antiviral response to replicate and propagate into their host. Rabies virus (RABV) phosphoprotein P is known to inhibit interferon induction. Here, using a global mass spectrometry approach, we show that RABV P binds to TBK1, a kinase located at the crossroads of many interferon induction pathways, resulting in innate immunity inhibition. Mutations of TBK1 phosphorylation sites abolish P binding. Importantly, we demonstrate that upon RABV infection or detection of dsRNA by innate immunity sensors, TBK1 and its adaptor proteins NAP1 and SINTBAD form dynamic cytoplasmic condensates that have liquid properties. These condensates can form larger aggregates having ring-like structures in which NAP1 and TBK1 exhibit locally restricted movement. P binding to TBK1 interferes with the formation of these structures. This work demonstrates that proteins of the signaling pathway leading to interferon induction transiently form liquid organelles that can be targeted by viruses. [Display omitted] • Upon interferon-inducing signals, TBK1, NAP1, and SINTBAD form liquid condensates • Rabies virus phosphoprotein P binds TBK1 to inhibit interferon induction pathways • A change of serine 179 to proline abolishes the P-inhibitory activity • Mutations of TBK1 phosphorylation sites abolish P binding Scrima et al. show that rabies virus (RABV) phosphoprotein P binds to phosphorylated TBK1, the kinase phosphorylating IRF3, and thus inhibits interferon induction. Upon RABV infection or dsRNA detection by innate immunity sensors, TBK1, NAP1, and SINTBAD form liquid condensates. The P-TBK1 association interferes with the formation of these condensates. [ABSTRACT FROM AUTHOR]

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL / BMAL1 . Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. [ABSTRACT FROM AUTHOR]

Wild grapevine ( Vitis vinifera subsp. sylvestris ) is the dioecious ancestral form of grapevine, from which the domesticated cultivars have derived ( V. vinifera subsp. vinifera ). Little is known about the floral scent compounds of wild grapevine that is considered as being partly insect pollinated. The knowledge of volatiles released by male and female inflorescence may contribute to the understanding of the pollination biology of this endangered taxon. Inflorescence scents of male and female individuals were collected by dynamic headspace and analysed by thermal desorption-GC/MS. A total of 17 compounds of C5-branched chain alcohols, aliphatics, aromatics, and terpenoids were identified with benzyl alcohol being most abundant in both sexes. Eight of the compounds were sex-specific and differences in 1,2-dimethoxybenzene were most obvious. This aromatic compound was a main constituent in the scent of females (30%), but it did not occur in males. Some of the main compounds of the scent samples are known to be detected by beetles (Cerambycidae) or attract sweat bees (Halictidae) and honey bees ( A. mellifera ), all well-known inflorescence visitors in wild grapevine. The data presented here are an important step in understanding the chemical communication between wild grapevine and its inflorescence visitors/potential pollinators. [ABSTRACT FROM AUTHOR]

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 BMAL1-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions. [ABSTRACT FROM AUTHOR]

Abstract: Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca2+; (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 Ca2+ 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. [Copyright &y& Elsevier]

Abstract: Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore an urgent need for a new generation of antifungal agents remains. We recently synthesised a set of linear and cyclic peptides characterized by sequences typical of membrane-active antimicrobial peptides (AMP). AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 () were tested against different yeast species and exhibited general antifungal activity, with a specificity against Cryptococcus neoformans. To evaluate the role of the membrane cell in the mechanism of antifungal activity, we investigated the conformational behaviour of AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 in different bio-membrane mimicking systems using a combined approach based on spectroscopy and microscopy techniques. Our data highlight the behaviour of the peptides to interact with the bilayer surface, excluding their ability to destabilize or permeabilize the fungal cell wall. Microbial membrane, indeed, may be an important platform for specific interactions of peptides with specific targets involved in the cell wall synthesis. [Copyright &y& Elsevier]

Abstract: Aβ (16–35) is the hydrophobic central core of β-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer''s disease patients. Depending on the conditions present, β-amyloid peptides undergo a conformational transition from random coil or α-helical monomers, to highly toxic β-sheet oligomers and aggregate fibrils. The behavior of β-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Aβ (16–35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles “doped” with small amounts of SDS. To analyze the tendency of Aβ (16–35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Aβ (16–35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Aβ (16–35) undergoes a conformational transition from a soluble helical–kink–helical structure, to a U-turn shaped conformation that resembles protofibril models. [Copyright &y& Elsevier]

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%; M age = 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. • As a proximal defense, fear of COVID-19 increases intention to get vaccinated. • Fear of COVID-19 increases existential anxiety. • Existential anxiety increases conspiracy beliefs. • Conspiracy beliefs increase intention to get vaccinated. • Existential anxiety and conspiracy beliefs mediate the relation between fear of COVID-19 and intention to get vaccinated. [ABSTRACT FROM AUTHOR]

HypB is a prokaryotic metal-binding guanine nucleotide-binding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Met hanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a com- mon feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ion-binding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases. [ABSTRACT FROM AUTHOR]

Abstract: The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtΔΨ, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C. [Copyright &y& Elsevier]

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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. Unlabelled Image • Synchronized HepG2 cells display autonomous rhythmic oscillation of OCR. • The AMP/ATP ratio undergoes oscillation in anti-phase with the rhythmic respiration. • PDH displays RR-sensitive rhythmic changes in the phosphorylation state. • OCR oscillation is sensitive to inhibitor of MCU, RyR Ca2+ Channel and ADPR cyclase. • RR treatment deregulates the rhythmic expression of circadian clock-related genes. [ABSTRACT FROM AUTHOR]

La fatigue est considérée comme chronique lorsqu'elle dure plus de 6 mois et qu'elle n'est pas améliorée par le repos. L'évaluation de la fatigue chronique (FC) reste difficile, les outils existants sont soit trop généraux, soit élaborés pour une pathologie spécifique. Associée ou non à une autre maladie chronique, elle constitue un phénomène complexe, multidimensionnel et subjectif expliquant le manque de consensus sur sa définition. L'objectif est d'explorer l'expérience des patients afin de construire une échelle d'autoévaluation de la FC. Trente et une personnes (neuf patients souffrant d'encéphalomyélite myalgique, douze patients ayant une maladie inflammatoire chronique de l'intestin et dix participants tout-venant) ont été interrogées sur la reconnaissance, la description et le vécu de leur fatigue. Leurs discours ont été étudiés par une analyse lexicométrique et une analyse phénoménologique interprétative. Les résultats ont montré des discours significativement différents entre les groupes cliniques et les participants tout-venant. En revanche, les deux groupes de patients présentent quatre dimensions communes : contraintes générées par la FC, conséquences physiques, conséquences sur la vie personnelle et conséquences sur les relations interpersonnelles. Ces dimensions suggèrent que le vécu des patients est plus complexe que ce qu'évaluent les outils existants. Ainsi, ces résultats plaident en faveur de l'élaboration d'une échelle d'évaluation de l'asthénie chronique construite à partir de l'expérience des participants. Asthenia is considered chronic when it extends over time for more than six months and remains unresolved by rest. Currently, chronic asthenia is difficult to assess as the existing scales are either too generic or, conversely, designed for asthenia associated with a specific pathology that hinders the correct evaluation of the associated fatigue. This is, however, problematic when it comes to chronic asthenia – whether or not it is associated with other chronic diseases – because it constitutes complex, multidimensional and largely subjective phenomena, all of which account for the lack of consensus in the literature about this condition. Therefore, any elaboration of a tool designed to evaluate fatigue in myalgic encephalomyelitis (ME) requires a thorough exploration of the patients' experience of this condition. This was precisely the main goal of the present study. Semi-structured interviews were collected from 31 individuals: a group of patients with ME (n = 9), a group of patients with inflammatory bowel disease (IBD, n = 12), and a group of control participants (n = 10), in which fourteen men with a mean age of 39.33 (±11.30) and seventeen women with a mean age of 43.5 (±12.8). The interviews focused on their perception of their relatives' fatigue and the description of their own fatigue. For the two groups of patients (ME, IBD), two additional themes were discussed: the evolution of their fatigue and their relationships with healthcare professionals. Two methods were used to analyze these interviews: lexicometric analysis (using IRaMuTeQ software) and an interpretative phenomenological analysis (IPA). This choice of combining quantitative and qualitative methods limits the subjectivity in discourse analysis. The lexicometric analysis revealed a significantly different content of discourse depending on the group of participants. The discourse of control participants focused on "normal" fatigue, which results from daily activities and can be resolved with rest. The IBD group emphasized the importance of the quality of sleep, the necessity of more frequent rest and fatigue-related pain. Finally, the discourse of participants in the ME group focused on the intensity of the manifestations of fatigue and the unpredictability of the need for rest which affects their everyday life. The interpretative phenomenological analysis revealed six major themes: origins of fatigue, different types of asthenias, its manifestations, its consequences, recovery time, and relationships with healthcare professionals. For all these themes, the discourses of ME group suggested a more intense and unpredictable fatigue than was the case for the two other groups. Taken together, the two types of analyses converged toward four common dimensions of fatigue that are experienced by the two groups of patients (as compared to the control group): constraints generated by chronic fatigue, consequences for their physical condition, their personal life, and their interpersonal relationships. These four dimensions appeared in the discourses of both patient groups, but the expressions about their fatigue and the relative impact were different. This study demonstrated that the experience of chronic asthenia is more complex than what can be assessed using existing specific tools, but also more specific than the dimensions of fatigue comprised in generic tools. These findings, therefore, confirmed the necessity for the developments of prospective assessment scales of chronic asthenia that take into account the patients' subjective experience of fatigue. [ABSTRACT FROM AUTHOR]

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 (3 h 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 Ca 2+ 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. [ABSTRACT FROM AUTHOR]

Bone diseases are medical conditions caused by the loss of bone homeostasis consecutive to increased osteoclast activity and diminished osteoblast activity. The mevalonate pathway (MVA) is crucial for maintaining this balance since it drives the post-translational prenylation of small guanosine triphosphatases (GTPases) proteins. Farnesyl pyrophosphate synthase (FPPS) plays a crucial role in the MVA pathway. Consequently, in the treatment of bone-related diseases, FPPS is the target of FDA-approved nitrogen-containing bisphosphonates (N-BPs), which have tropism mainly for bone tissue due to their poor penetration in soft tissues. The development of inhibitors targeting the FPPS enzyme has garnered significant interest in recent decades due to FPPS's role in the biosynthesis of cholesterol and other isoprenoids, which are implicated in cancer, bone diseases, and other conditions. In this study, we describe a multidisciplinary approach to designing novel FPPS inhibitors, combining computational modeling, biochemical assays, and biophysical techniques. A series of peptides and phosphopeptides were designed, synthesized, and evaluated for their ability to inhibit FPPS activity. Molecular docking was employed to predict the binding modes of these compounds to FPPS, while Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) spectroscopy experiments – based on Saturation Transfer Difference (STD) and an enzymatic NMR assay – were used to measure their binding affinities and kinetics. The biological activity of the most promising compounds was further assessed in cellular assays using murine colorectal cancer (CRC) cells. Additionally, genomics and metabolomics profiling allowed to unravel the possible mechanisms underlying the activity of the peptides, confirming their involvement in the modulation of the MVA pathway. Our findings demonstrate that the designed peptides and phosphopeptides exhibit significant inhibitory activity against FPPS and possess antiproliferative effects on CRC cells, suggesting their potential as therapeutic agents for cancer. [Display omitted] • FPPS is a key enzyme in the mevalonate (MVA) pathway and is the target of antitumoral FDA-approved molecules. • Here we present the design, synthesis, and evaluation of pentapeptides and their derivatives as potential ligands of FPPS. • The peptides showed antiproliferative effect on murine colon adenocarcinoma cells and the ability to interfere with the MVA pathway, suggesting a potential selectivity for FPPS. [ABSTRACT FROM AUTHOR]

Abstract: 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) (HgCl2) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl2 as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl2-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 HgCl2-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 HgCl2-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming. [Copyright &y& Elsevier]

Abstract: 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 ΔΨm-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. [Copyright &y& Elsevier]

Abstract: The metabolic control analysis was applied to digitonin-permeabilized HepG2 cell line to assess the flux control exerted by cytochrome c oxidase on the mitochondrial respiration. Experimental conditions eliciting different energy/respiratory states in mitochondria were settled. The results obtained show that the mitochondrial electrochemical potential accompanies a depressing effect on the control coefficient exhibited by the cytochrome c oxidase. Both the components of the protonmotive force, i.e. the voltage (ΔΨm) and the proton (ΔpHm) gradient, displayed a similar effect. Quantitative estimation of the ΔΨm unveiled that the voltage-dependent effect on the control coefficient of cytochrome c oxidase takes place sharply in a narrow range of membrane potential from 170–180 to 200–210mV consistent with the physiologic transition from state 3 to state 4 of respiration. Extension of the metabolic flux control analysis to the NADH dehydrogenase and bc1 complexes of the mitochondrial respiratory chain resulted in a similar effect. A mechanistic model is put forward whereby the respiratory chain complexes are proposed to exist in a voltage-mediated threshold-controlled dynamic equilibrium between supercomplexed and isolated states. [Copyright &y& Elsevier]

Abstract: This study aimed to validate, in situ, proposed mechanisms of bupivacaine citotoxicity 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 mtΔμ H+-controlled respiratory rates in a cyclosporine A-insensitive manner and further inhibition of the respiratory rates. Intriguingly, a relatively small decrease on the mtΔΨ (about 20mV) 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. [ABSTRACT FROM AUTHOR]

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 Å resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three α-helices. The active methyltransferase is the heterodimer Ydr140w·Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold. [ABSTRACT FROM AUTHOR]

This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polar-ographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the [This symbol cannot be presented in ASCII format] producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells. [ABSTRACT FROM AUTHOR]

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 -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 withVH3/VK1variable 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. Insummary,ourresults provide extensive insights into the structure of InvD and its specific interaction with the target molecule in the host. [ABSTRACT FROM AUTHOR]

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis inducing protein (DAIP). The amino acid sequence of DAIP contains five potential glutamines and ten lysines for MTG mediated cross-linking. Aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in E. coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four out of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed preference of MTG for the DAIP glutamines in the order of Q39>>Q298>Q345~Q65>>Q144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and the structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity and efficiency. [ABSTRACT FROM AUTHOR]

Abstract: Analogs of potent CaMKinase II inhibitor, CaM-KNtide, were prepared to explore new structural requirements for the inhibitory activity. The full potency of CaMKII inhibition by CaM-KIINα is contained within a minimal region of 19 amino acids. Here, analysis of the homologous CaM-KIINβ showed that a 17 mer peptide (CN17β) was the shortest sequence that still retained useful inhibitory potency. Ala substitution of almost any residue of CN17β dramatically reduced potency, except for substitution of P3, R14, and V16. Fusion with the tat sequence generated the cell-penetrating inhibitor version tat-5. This tat-5 fusion peptide maintained selectivity for CaMKII over CaMKI and CaMKIV, and appeared to slightly further enhance potency (IC50 ∼30 nM). Within a breast cancer cell line and in primary human fibroblasts, tat-5 inhibited the Erk signaling pathway and proliferation without any measurable cytotoxicity. Structural analysis of CN17β by CD and NMR indicated an α-helix conformation in the Leu6-Arg11 segment well overlapping with the crystal structure of 21-residue segment of CaM-KNtide bound to the kinase domain of CaMKII. [Copyright &y& Elsevier]

Abstract: Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans. [ABSTRACT FROM AUTHOR]

Abstract: Cells infected by the hepatitis C virus (HCV) are characterized by endoplasmic reticulum stress, deregulation of the calcium homeostasis and unbalance of the oxido-reduction state. In this context, mitochondrial dysfunction proved to be involved and is thought to contribute to the outcome of the HCV-related disease. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca2+ from the endoplasmic reticulum, followed by uptake into mitochondria. This causes successive mitochondrial alterations comprising generation of reactive oxygen and nitrogen species and impairment of the oxidative phosphorylation. A progressive adaptive response results in an enhancement of the glycolytic metabolism sustained by up-regulation of the hypoxia inducible factor. Pathogenetic implications of the model are discussed. [Copyright &y& Elsevier]

A short and divergent route towards new derivatives of 2-(trifluoromethyl)pyridines as potent inverse agonists of the bacterial target PqsR against Pseudomonas aeruginosa (PA) infections is described. This Gram-negative pathogen causes severe nosocomial infections and common antibiotic treatment options are rendered ineffective due to resistance issues. Based on an earlier identified optimized hit, we conducted derivatization and rigidification attempts employing two central building blocks. The western part of the molecule is built up via a 2-(trifluoromethyl)pyridine head group equipped with a terminal alkyne. The eastern section is then introduced through aryliode motifs exploiting Sonogashira as well as Suzuki-type chemistry. Subsequent modification provided quick access to an array of compounds, allowed for deep SAR insights, and enabled to optimize the hit scaffold into a lead structure of nanomolar potency combined with favorable in vitro ADME/T features. [Display omitted] • Divergency-oriented synthesis of Quorum Sensing inhibitors (QSI). • Successful hit-to-lead multiparameter optimization. • Novel QSI show favorable DMPK properties and safety pharmacology. • Co–crystallization of a QSI in complex with target protein. • Significant effect on Pseudomonas aeruginosa biofilm formation with Tobramycin. [ABSTRACT FROM AUTHOR]

Abstract: Bradykinin (BK) is involved in a wide variety of pathophysiological processes. Potent BK peptide antagonists can be developed introducing constrained unnatural amino acids, necessary to force the secondary structure of the molecule. In this paper, we report a structure–activity relationship study of two peptide analogues of the potent B2 antagonist HOE 140 by replacing the D-Tic-Oic dipeptide with conformationally constrained dipeptide mimetic β-turn inducers. [Copyright &y& Elsevier]