NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

A non-circadian role for clock-genes in sleep homeostasis: a strain comparison.

BACKGROUND: We have previously reported that the expression of circadian clock-genes increases in the cerebral cortex after sleep deprivation (SD) and that the sleep rebound following SD is attenuated in mice deficient for one or more clock-genes. We hypothesized that besides generating circadian rhythms, clock-genes also play a role in the homeostatic regulation of sleep. Here we follow the time course of the forebrain changes in the expression of the clock-genes period (per)-1, per2, and of the clock-controlled gene albumin D-binding protein (dbp) during a 6 h SD and subsequent recovery sleep in three inbred strains of mice for which the homeostatic sleep rebound following SD differs. We reasoned that if clock genes are functionally implicated in sleep homeostasis then the SD-induced changes in gene expression should vary according to the genotypic differences in the sleep rebound. RESULTS: In all three strains per expression was increased when animals were kept awake but the rate of increase during the SD as well as the relative increase in per after 6 h SD were highest in the strain for which the sleep rebound was smallest; i.e., DBA/2J (D2). Moreover, whereas in the other two strains per1 and per2 reverted to control levels with recovery sleep, per2 expression specifically, remained elevated in D2 mice. dbp expression increased during the light period both during baseline and during SD although levels were reduced during the latter condition compared to baseline. In contrast to per2, dbp expression reverted to control levels with recovery sleep in D2 only, whereas in the two other strains expression remained decreased. CONCLUSION: These findings support and extend our previous findings that clock genes in the forebrain are implicated in the homeostatic regulation of sleep and suggest that sustained, high levels of per2 expression may negatively impact recovery sleep.

Whole-heart coronary vein imaging: a comparison between non-contrast-agent- and contrast-agent-enhanced visualization of the coronary venous system.

The feasibility of three-dimensional (3D) whole-heart imaging of the coronary venous (CV) system was investigated. The hypothesis that coronary magnetic resonance venography (CMRV) can be improved by using an intravascular contrast agent (CA) was tested. A simplified model of the contrast in T(2)-prepared steady-state free precession (SSFP) imaging was applied to calculate optimal T(2)-preparation durations for the various deoxygenation levels expected in venous blood. Non-contrast-agent (nCA)- and CA-enhanced images were compared for the delineation of the coronary sinus (CS) and its main tributaries. A quantitative analysis of the resulting contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) in both approaches was performed. Precontrast visualization of the CV system was limited by the poor CNR between large portions of the venous blood and the surrounding tissue. Postcontrast, a significant increase in CNR between the venous blood and the myocardium (Myo) resulted in a clear delineation of the target vessels. The CNR improvement was 347% (P < 0.05) for the CS, 260% (P < 0.01) for the mid cardiac vein (MCV), and 430% (P < 0.05) for the great cardiac vein (GCV). The improvement in SNR was on average 155%, but was not statistically significant for the CS and the MCV. The signal of the Myo could be significantly reduced to about 25% (P < 0.001).

Ultraschalldiagnostik des nichttraumatischen akuten Abdomens: die Ursachen ausserhalb des Gastro-Intestinaltraktes [Ultrasound of non traumatic acute abdomen: extra-digestive causes]

In recent years, management of abdominal pain in emergency care units (ECU) has undergone a radical change. Chronic work overload of ECU and increase in severity and complexity of cases required an optimal medical imaging method, such as ultrasound, to establish an accurate diagnosis as quickly as possible. A great variety of causes may cause acute abdominal pain and the role of sonography is to accurately distinguish between these aetiologies. For the application of sonography, perhaps more so than with other imaging methods, a perfect knowledge of sonographical characteristics and technical possibilities is essential to achieve an optimal answer. The purpose of this review is to present and discuss the sonographic characteristics of extra-digestive causes of acute abdominal pain such as splenic infarction, thoracic pathologies, urinary and gynaecologic diseases or retroperitoneal pathologies.

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Review of the Peri-operative Non-medical Workforce in the HPSS (PDF 720 KB)

A Review of the Peri-operative Non Medical Workforce within Northern Ireland Encompassing the Twelve Acute Trusts

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P &lt; 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

Non-nest mate discrimination and clonal colony structure in the parthenogenetic ant Cerapachys biroi

Understanding the interplay between cooperation and conflict in social groups is a major goal of biology. One important factor is genetic relatedness, and animal societies are usually composed of related but genetically different individuals, setting the stage for conflicts over reproductive allocation. Recently, however, it has been found that several ant species reproduce predominantly asexually. Although this can potentially give rise to clonal societies, in the few well-studied cases, colonies are often chimeric assemblies of different genotypes, due to worker drifting or colony fusion. In the ant Cerapachys biroi, queens are absent and all individuals reproduce via thelytokous parthenogenesis, making this species an ideal study system of asexual reproduction and its consequences for social dynamics. Here, we show that colonies in our study population on Okinawa, Japan, recognize and effectively discriminate against foreign workers, especially those from unrelated asexual lineages. In accord with this finding, colonies never contained more than a single asexual lineage and average pairwise genetic relatedness within colonies was extremely high (r = 0.99). This implies that the scope for social conflict in C. biroi is limited, with unusually high potential for cooperation and altruism.

Profitability, uncertainty and multi-product firm product proliferation: The Spanish car industry

This article studies how product introduction decisions relate to profitability and uncertainty in the context of multi-product firms and product differentiation. These two features, common to many modern industries, have not received much attention in the literature as compared to the classical problem of firm entry, even if the determinants of firm and product entry are quite different. The theoretical predictions about the sign of the impact of uncertainty on product entry are not conclusive. Therefore, an econometric model relating firms’ product introduction decisions with profitability and profit uncertainty is proposed. Firm’s estimated profits are obtained from a structural model of product demand and supply, and uncertainty is proxied by profits’ variance. The empirical analysis is carried out using data on the Spanish car industry for the period 1990-2000. The results show a positive relationship between product introduction and profitability, and a negative one with respect to profit variability. Interestingly, the degree of uncertainty appears to be a driving force of entry stronger than profitability, suggesting that the product proliferation process in the Spanish car market may have been mainly a consequence of lower uncertainty rather than the result of having a more profitable market. Keywords: Product introduction, entry, uncertainty, multiproduct firms, automobile JEL codes: L11, L13

Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Vertebrate conserved non coding DNA regions have a high persistence length and a short persistence time.

BACKGROUND: The comparison of complete genomes has revealed surprisingly large numbers of conserved non-protein-coding (CNC) DNA regions. However, the biological function of CNC remains elusive. CNC differ in two aspects from conserved protein-coding regions. They are not conserved across phylum boundaries, and they do not contain readily detectable sub-domains. Here we characterize the persistence length and time of CNC and conserved protein-coding regions in the vertebrate and insect lineages. RESULTS: The persistence length is the length of a genome region over which a certain level of sequence identity is consistently maintained. The persistence time is the evolutionary period during which a conserved region evolves under the same selective constraints.Our main findings are: (i) Insect genomes contain 1.60 times less conserved information than vertebrates; (ii) Vertebrate CNC have a higher persistence length than conserved coding regions or insect CNC; (iii) CNC have shorter persistence times as compared to conserved coding regions in both lineages. CONCLUSION: Higher persistence length of vertebrate CNC indicates that the conserved information in vertebrates and insects is organized in functional elements of different lengths. These findings might be related to the higher morphological complexity of vertebrates and give clues about the structure of active CNC elements.Shorter persistence time might explain the previously puzzling observations of highly conserved CNC within each phylum, and of a lack of conservation between phyla. It suggests that CNC divergence might be a key factor in vertebrate evolution. Further evolutionary studies will help to relate individual CNC to specific developmental processes.