Osmotic stress tolerance mechanisms in Lactococcus lactis

The response of Lactococcus lactis subsp. cremoris NCDO 712 to low water activity (aw) was investigated, both in relation to growth following moderate reductions in the aw and in terms of survival following substantial reduction of the aw with NaCI. Lc.lactis NCDO 712 was capable of growth in the presence of ≤ 4% w/v NaCI and concentrations in excess of 4% w/v were lethal to the cells. The presence of magnesium ions significantly increased the resistance of NCDO 712 to challenge with NaCI and also to challenge with high temperature or low pH. Survival of Lc.lactis NCDO 712 exposed to high NaCI concentrations was growth phase dependent and cells were most sensitive in the early exponential phase of growth. Pre-exposure to 3% w/v NaCI induced limited protection against subsequent challenge with higher NaCI concentrations. The induction was inhibited by chloramphenicol and even when induced, the response did not protect against NaCI concentrations> 10% w/v. When growing at low aw, potassium was accumulated by Lc. lactis NCDO 712 growing at low aw, if the aw was reduced by glucose or fructose, but not by NaCI. Reducing the potassium concentration of chemically defined medium from 20 to 0.5 mM) produced a substantial reduction in the growth rate, if the aw was reduced with NaCI, but not with glucose or fructose. The reduction of the growth rate correlated strongly with a reduction in the cytoplasmic potassium concentration and in cell volume. Addition of the compatible solute glycine betaine, partially reversed the inhibition of growth rate and partially restored the cell volume. The potassium transport system was characterised in cells grown in medium at both high and low aw. It appeared that a single system was present, which was induced approximately two-fold by growth at low aw. Potassium transport was assayed in vitro using cells depleted of potassium; the assay was competitively inhibited by Na+ and by the other monovalent cations NH4+, Li+, and Cs+. There was a strong correlation between the ability of strains of Lc. lactis subsp. lactis and subsp. cremoris to grow at low aw and their ability to accumulate the compatible solute glycine betaine. The Lc. lactis subsp. cremoris strains incapable of growth at NaCI concentrations> 2% w/v did not accumulate glycine betaine when growing at low aw, whereas strains capable of growth at NaCI concentrations up to 4% w/v did. A mutant, extremely sensitive to low aw was isolated from the parent strain Lc. lactis subsp. cremoris MG 1363, a plasmid free derivative of NCDO 712. The parent strain tolerated up to 4% w/v NaCI and actively accumulated glycine betaine when challenged at low aw. The mutant had lost the ability to accumulate glycine betaine and was incapable of growth at NaCI concentrations >2% w/v or the equivalent concentration of glucose. As no other compatible solute seemed capable of substitution for glycine betaine, the data suggest that the traditional; phenotypic speciation of strains on the basis of tolerance to 4% w/v NaCI can be explained as possession or lack of a glycine betaine transport system.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Usability-enhanced coordination design of geovisualisations to communicate coastal flood risk information

For at least two millennia and probably much longer, the traditional vehicle for communicating geographical information to end-users has been the map. With the advent of computers, the means of both producing and consuming maps have radically been transformed, while the inherent nature of the information product has also expanded and diversified rapidly. This has given rise in recent years to the new concept of geovisualisation (GVIS), which draws on the skills of the traditional cartographer, but extends them into three spatial dimensions and may also add temporality, photorealistic representations and/or interactivity. Demand for GVIS technologies and their applications has increased significantly in recent years, driven by the need to study complex geographical events and in particular their associated consequences and to communicate the results of these studies to a diversity of audiences and stakeholder groups. GVIS has data integration, multi-dimensional spatial display advanced modelling techniques, dynamic design and development environments and field-specific application needs. To meet with these needs, GVIS tools should be both powerful and inherently usable, in order to facilitate their role in helping interpret and communicate geographic problems. However no framework currently exists for ensuring this usability. The research presented here seeks to fill this gap, by addressing the challenges of incorporating user requirements in GVIS tool design. It starts from the premise that usability in GVIS should be incorporated and implemented throughout the whole design and development process. To facilitate this, Subject Technology Matching (STM) is proposed as a new approach to assessing and interpreting user requirements. Based on STM, a new design framework called Usability Enhanced Coordination Design (UECD) is ten presented with the purpose of leveraging overall usability of the design outputs. UECD places GVIS experts in a new key role in the design process, to form a more coordinated and integrated workflow and a more focused and interactive usability testing. To prove the concept, these theoretical elements of the framework have been implemented in two test projects: one is the creation of a coastal inundation simulation for Whitegate, Cork, Ireland; the other is a flooding mapping tool for Zhushan Town, Jiangsu, China. The two case studies successfully demonstrated the potential merits of the UECD approach when GVIS techniques are applied to geographic problem solving and decision making. The thesis delivers a comprehensive understanding of the development and challenges of GVIS technology, its usability concerns, usability and associated UCD; it explores the possibility of putting UCD framework in GVIS design; it constructs a new theoretical design framework called UECD which aims to make the whole design process usability driven; it develops the key concept of STM into a template set to improve the performance of a GVIS design. These key conceptual and procedural foundations can be built on future research, aimed at further refining and developing UECD as a useful design methodology for GVIS scholars and practitioners.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.

Stress-induced visceral pain in rodents: neurochemical, hormonal, immune and epigenetic mechanisms

Visceral pain is a debilitating disorder which affects up to 25% of the population at any one time. It is a global term used to describe pain originating from the internal organs, which is distinct from somatic pain. Currently the treatment strategies are unsatisfactory, with development of novel therapeutics hindered by a lack of detailed knowledge of the underlying mechanisms. The work presented in this thesis aimed to redress this issue and look in more detail at the molecular mechanisms of visceral pain in preclinical models. Stress has long been implicated in the pathophysiology of visceral pain in both preclinical and clinical studies. Here a mouse model of early-life stress-induced visceral hypersensitivity was validated. Moreover, mouse strain differences were also apparent in visceral sensitivity suggesting a possible genetic component to the underlying pathophysiology. Furthermore, gender and sex hormones were also implicated in stress sensitivity and visceral pain. Using the rat model of maternal separation, some of the epigenetic mechanisms underpinning visceral hypersensitivity, specifically the contribution of histone acetylation were unravelled. Glutamate has been well established in somatic pain processing, however, its contribution to visceral pain has not been extensively characterised. It was found that glutamate uptake is impaired in viscerally hypersensitive animals, an effect which could be reversed by treatment with riluzole, a glutamate uptake activator. Moreover, negative modulation of the metabotropic glutamate (mGlu) receptor 7 was sufficient to reverse visceral hypersensitivity in a stress sensitive rat strain, the Wistar Kyoto rat. Furthermore, toll-like receptor 4 (TLR4) was implicated in chronic stress-induced visceral hypersensitivity. Taken together, these findings have furthered our knowledge of the pathophysiology of visceral pain. In addition, we have identified glutamate transporters, mGlu7 receptor, histone acetylation and TLR4 as novel targets, amenable to pharmacological manipulation for the specific treatment of visceral pain.

Molecular analysis of inflammatory mechanisms associated with Escherichia coli and inflammatory bowel diseases

Inflammatory bowel diseases (IBD), encompasses a range of chronic, immune-mediated inflammatory disorders that are usually classified under two major relapsing conditions, Crohn’s Disease (CD) and ulcerative colitis (UC). Extensive studies in the last decades have suggested that the etiology of IBD involves environmental and genetic factors that lead to dysfunction of epithelial barrier with consequent deregulation of the mucosal immune system and inadequate responses to gut microbiota.Over the last decade, the microbial species that has attracted the most attention, with respect to CD etiology, is Eschericia coli. In CD tissue, E. coli antigens have also been identified in macrophages within the lamina propria, granulomas, and in the germinal centres of mesenteric lymph nodes of patients. They have been shown to adhere to and invade intestinal epithelial cells whilst also being able to extensively replicate within macrophages. Through the work of genome-wide association studies (GWAS), there is growing evidence to suggest that the microbial imbalance between commensal and pathogenic bacteria in the gut is aided by a defect in the innate immune system. Autophagy represents a recently investigated pathway that is believed to contribute to the pathogenesis of CD, with studies identified a variant of the autophagy gene, ATG16L1, as a susceptibility gene. The aim of my thesis was to study the cellular and molecular mechanism promoted by E.coli strains in epithelial cells and to assess their contribution to IBD pathology. To achieve this we focused on developing both an in vitro and in vivo model of AIEC infection. This allowed us to further our knowledge on possible mechanisms utilised by AIEC that promoted their survival, as well as developing a better understanding of host reactions. We demonstrate a new survival mechanism promoted by E.coli HM605, whereby it induces the expression of the anti-apoptotic proteins Bcl-XL and BCL2, all of which is exacerbated in an autophagy deficient system. We have also demonstrated the presence of AIEC-induced inflammasome responses in epithelial cells which are exacerbated in an autophagy deficient system and expression of NOD-like receptors (NLRs) which might mediate inflammasome responses in vivo. Finally, we used the Citrobacter rodentium model of infectious colitis to identify Pellino3 as an important mediator in the NOD2 pathway and regulator of intestinal inflammation. In summary, we have developed robust and versatile models of AIEC infection as well as provide new insights into AIEC mediated survival pathways. The collected data provides a new perception into why AIEC bacteria are able to prosper in conditions associated with Crohn’s disease patients with a defect in autophagy.

Identification and analysis of mechanisms involved in a broad-spectrum disease resistant mutant of the winter wheat cv. Guardian

M66 an X-ray induced mutant of winter wheat (Triticum aestivum) cv. Guardian exhibits broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. tritici), yellow rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia recondita f. sp. tritici), along with partial resistance to stagnonospora nodorum blotch (caused by the necrotroph Stagonosporum nodorum) and septoria tritici blotch (caused by the hemibiotroph Mycosphaerella graminicola) compared to the parent plant ‘Guardian’. Analysis revealed that M66 exhibited no symptoms of infection following artificial inoculation with Bgt in the glasshouse after adult growth stage (GS 45). Resistance in M66 was associated with widespread leaf flecking which developed during tillering. Flecking also occurred in M66 leaves without Bgt challenge; as a result grain yields were reduced by approximately 17% compared to ‘Guardian’ in the absence of disease. At the seedling stage, M66 exhibited partial resistance. M66, along with Tht mutants (Tht 12, Tht13), also exhibit increased tolerance to environmental stresses (abiotic), such as drought and heat stress at seedling and adult growth stages, However, adult M66 exhibited increased susceptibility to the aphid Schizaphis graminum compared to ‘Guardian’. Resistance to Bgt in M66 was characterized with increased and earlier H2O2 accumulation at the site of infection which resulted in increased papilla formation in epidermal cells, compared to ‘Guardian’. Papilla formation was associated with reduced pathogen ingress and haustorium formation, indicating that the primary cause of resistance in M66 was prevention of pathogen penetration. Heat treatment at 46º C prior to challenge with Bgt also induced partial disease resistance to Blumeria graminis f. sp. tritici in ‘Guardian’ and M66 seedlings. This was characterized by a delay in primary infection, due to increased production of ROS species, such as hydrogen peroxide, ROS-scavenging enzymes and Hsp70, resulting in cross-linking of cell wall components prior to inoculation. This actively prevented the fungus from penetrating the epidermal cell wall. Proteomics analysis using 2-D gel electrophoresis identified primary and secondary disease resistance effects in M66 including detection of ROS scavenging enzymes (4, 24 hai), such as ascorbate peroxidase and a superoxidase dismutase isoform (CuZnSOD) in M66 which were absent from ‘Guardian’. Chitinase (PR protein) was also upregulated (24 hai) in M66 compared to ‘Guardian’.Monosomic and ditelosomic analysis of M66 revealed that the mutation in M66 is located on the long arm of chromosome 2B (2BL). Chromosome 2BL is known to have key genes involved in resistance to pathogens such as those causing stripe rust and powdery mildew. The TaMloB1 gene, an orthologue of the barley Mlo gene, is also located on chromosome 2BL. Sanger sequencing of part of the coding sequence revealed no deletions in the TaMloB1 gene between ‘Guardian’ and M66.

Molecular mechanisms underpinning IFN-gamma and TNF-alpha synergy in intestinal epithelial cells

Cytokine-driven signalling shapes immune homeostasis and guides inflammatory responses mainly through induction of specific gene expression programmes both within and outside the immune cell compartment. These transcriptional outputs are often amplified via cytokine synergy, which sets a stimulatory threshold that safeguards from exacerbated inflammation and immunopathology. In this study, we investigated the molecular mechanisms underpinning synergy between two pivotal Th1 cytokines, IFN-γ and TNF-α, in human intestinal epithelial cells. These two proinflammatory mediators induce a unique state of signalling and transcriptional synergy implicated in processes such as antiviral and antitumour immunity, intestinal barrier and pancreatic β-cell dysfunction. Since its discovery more than 30 years ago, this biological phenomenon remains, however, only partially defined. Here, using a functional genomics approach including RNAi perturbation screens and small-molecule inhibitors, we identified two new regulators of IFN-γ/TNF-α-induced chemokine and antiviral gene and protein expression, a Bcl-2 protein BCL-G and a histone demethylase UTX. We also discovered that IFN-γ/TNF-α synergise to trigger a coordinated shutdown of major receptor tyrosine kinases expression in colon cancer cells. Together, these findings extend our current understanding of how IFN-γ/TNF-α synergy elicits qualitatively and quantitatively distinct outputs in the intestinal epithelium. Given the well-documented role of this synergistic state in immunopathology of various disorders, our results may help to inform the identification of high quality and biologically relevant druggable targets for diseases characterised by an IFN-γ/TNF-α high immune signature

Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity

The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.

Safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon Alfa-2a in HIV-1-monoinfected participants: a phase II clinical trial.

BACKGROUND: To our knowledge, the antiviral activity of pegylated interferon alfa-2a has not been studied in participants with untreated human immunodeficiency virus type 1 (HIV-1) infection but without chronic hepatitis C virus (HCV) infection. METHODS: Untreated HIV-1-infected volunteers without HCV infection received 180 microg of pegylated interferon alfa-2a weekly for 12 weeks. Changes in plasma HIV-1 RNA load, CD4(+) T cell counts, pharmacokinetics, pharmacodynamic measurements of 2',5'-oligoadenylate synthetase (OAS) activity, and induction levels of interferon-inducible genes (IFIGs) were measured. Nonparametric statistical analysis was performed. RESULTS: Eleven participants completed 12 weeks of therapy. The median plasma viral load decrease and change in CD4(+) T cell counts at week 12 were 0.61 log(10) copies/mL (90% confidence interval [CI], 0.20-1.18 log(10) copies/mL) and -44 cells/microL (90% CI, -95 to 85 cells/microL), respectively. There was no correlation between plasma viral load decreases and concurrent pegylated interferon plasma concentrations. However, participants with larger increases in OAS level exhibited greater decreases in plasma viral load at weeks 1 and 2 (r = -0.75 [90% CI, -0.93 to -0.28] and r = -0.61 [90% CI, -0.87 to -0.09], respectively; estimated Spearman rank correlation). Participants with higher baseline IFIG levels had smaller week 12 decreases in plasma viral load (0.66 log(10) copies/mL [90% CI, 0.06-0.91 log(10) copies/mL]), whereas those with larger IFIG induction levels exhibited larger decreases in plasma viral load (-0.74 log(10) copies/mL [90% CI, -0.93 to -0.21 log(10) copies/mL]). CONCLUSION: Pegylated interferon alfa-2a was well tolerated and exhibited statistically significant anti-HIV-1 activity in HIV-1-monoinfected patients. The anti-HIV-1 effect correlated with OAS protein levels (weeks 1 and 2) and IFIG induction levels (week 12) but not with pegylated interferon concentrations.

Competing manufacturers in a retail supply chain: On contractual form and coordination

It is common for a retailer to sell products from competing manufacturers. How then should the firms manage their contract negotiations? The supply chain coordination literature focuses either on a single manufacturer selling to a single retailer or one manufacturer selling to many (possibly competing) retailers. We find that some key conclusions from those market structures do not apply in our setting, where multiple manufacturers sell through a single retailer. We allow the manufacturers to compete for the retailer's business using one of three types of contracts: a wholesale-price contract, a quantity-discount contract, or a two-part tariff. It is well known that the latter two, more sophisticated contracts enable the manufacturer to coordinate the supply chain, thereby maximizing the profits available to the firms. More importantly, they allow the manufacturer to extract rents from the retailer, in theory allowing the manufacturer to leave the retailer with only her reservation profit. However, we show that in our market structure these two sophisticated contracts force the manufacturers to compete more aggressively relative to when they only offer wholesale-price contracts, and this may leave them worse off and the retailer substantially better off. In other words, although in a serial supply chain a retailer may have just cause to fear quantity discounts and two-part tariffs, a retailer may actually prefer those contracts when offered by competing manufacturers. We conclude that the properties a contractual form exhibits in a one-manufacturer supply chain may not carry over to the realistic setting in which multiple manufacturers must compete to sell their goods through the same retailer. © 2010 INFORMS.

C. elegans germline-deficient mutants respond to pathogen infection using shared and distinct mechanisms.

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including gram positive and gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16.

MicroRNA antagonism of the picornaviral life cycle: alternative mechanisms of interference.

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3' untranslated region (3'UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.

Neural mechanisms of context effects on face recognition: automatic binding and context shift decrements.

Although people do not normally try to remember associations between faces and physical contexts, these associations are established automatically, as indicated by the difficulty of recognizing familiar faces in different contexts ("butcher-on-the-bus" phenomenon). The present fMRI study investigated the automatic binding of faces and scenes. In the face-face (F-F) condition, faces were presented alone during both encoding and retrieval, whereas in the face/scene-face (FS-F) condition, they were presented overlaid on scenes during encoding but alone during retrieval (context change). Although participants were instructed to focus only on the faces during both encoding and retrieval, recognition performance was worse in the FS-F than in the F-F condition ("context shift decrement" [CSD]), confirming automatic face-scene binding during encoding. This binding was mediated by the hippocampus as indicated by greater subsequent memory effects (remembered > forgotten) in this region for the FS-F than the F-F condition. Scene memory was mediated by right parahippocampal cortex, which was reactivated during successful retrieval when the faces were associated with a scene during encoding (FS-F condition). Analyses using the CSD as a regressor yielded a clear hemispheric asymmetry in medial temporal lobe activity during encoding: Left hippocampal and parahippocampal activity was associated with a smaller CSD, indicating more flexible memory representations immune to context changes, whereas right hippocampal/rhinal activity was associated with a larger CSD, indicating less flexible representations sensitive to context change. Taken together, the results clarify the neural mechanisms of context effects on face recognition.