Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.     

The Effect of Divalent Cations and Core Polymerase in the Abortive Initiation by Escherichia Coli Dna-Dependent Rna Polymerase Using Phosphonate Dinucleotide Primers

The effect of divalent Mg²⁺ and Mn²⁺ cations on the elongation of ApU, UpA and their 3'-O- and 5'-O-phosphonylmethyl analogues by RNA polymerase holoenzyme to the corresponding trinucleo-tides on a poly(dA-dT) template was investigated. In contrast to Mg^z+ ions, Mn²⁺ ions enhance abortive trinucleotide synthesis. This effect is more pronounced with phosphonylmethyl analogues. The core enzyme cannot catalyze the elongation of either (2'-5') UpA or phosphonylmethyl analogues. The localization of the divalent cation activator, as well as the role of the σ subunit at the catalytic centre of the holoenzyme, is discussed.     

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.     

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Fabrication of Dense Non-Circular Nanomagnetic Device Arrays Using Self-Limiting Low-Energy Glow-Discharge Processing

We describe a low-energy glow-discharge process using reactive ion etching system that enables non-circular device patterns, such as squares or hexagons, to be formed from a precursor array of uniform circular openings in polymethyl methacrylate, PMMA, defined by electron beam lithography. This technique is of a particular interest for bit-patterned magnetic recording medium fabrication, where close packed square magnetic bits may improve its recording performance. The process and results of generating close packed square patterns by self-limiting low-energy glow-discharge are investigated. Dense magnetic arrays formed by electrochemical deposition of nickel over self-limiting formed molds are demonstrated.     

Mechanisms and Consequences of Developmental Acceleration in Tadpoles Responding to Pond Drying

Many amphibian species exploit temporary or even ephemeral aquatic habitats for reproduction by maximising larval growth under benign conditions but accelerating development to rapidly undergo metamorphosis when at risk of desiccation from pond drying. Here we determine mechanisms enabling developmental acceleration in response to decreased water levels in western spadefoot toad tadpoles (Pelobates cultripes), a species with long larval periods and large size at metamorphosis but with a high degree of developmental plasticity. We found that P. cultripes tadpoles can shorten their larval period by an average of 30% in response to reduced water levels. We show that such developmental acceleration was achieved via increased endogenous levels of corticosterone and thyroid hormone, which act synergistically to achieve metamorphosis, and also by increased expression of the thyroid hormone receptor TRΒ, which increases tissue sensitivity and responsivity to thyroid hormone. However, developmental acceleration had morphological and physiological consequences. In addition to resulting in smaller juveniles with proportionately shorter limbs, tadpoles exposed to decreased water levels incurred oxidative stress, indicated by increased activity of the antioxidant enzymes catalase, superoxide dismutase, and gluthatione peroxidase. Such increases were apparently sufficient to neutralise the oxidative damage caused by presumed increased metabolic activity. Thus, developmental acceleration allows spadefoot toad tadpoles to evade drying ponds, but it comes at the expense of reduced size at metamorphosis and increased oxidative stress.     

SIRT1 is involved in adrenocortical cancer growth and motility

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF-II (insulin-like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up-regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.     

Genetic Diversity and Structure of Dalmatian Pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip., Asteraceae) within the Balkan Refugium

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.     

Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes

The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla–firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.