Amino acids 1-20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES- dependent translation in Hep G2 cells, and inhibit both HCV IRES- and cap-dependent translation in HuH7 and CV-1 cells.

A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1-20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia-HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1-20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NES1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint.

Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice

Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody- mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.

Using the flexibility index to compare batch and continuous activated sludge processes

This paper considers the question of which is better: the batch or the continuous activated sludge processes? It is an important question because dissension still exists in the wastewater industry as to the relative merits of each of the processes. A review of perceived differences in the processes from the point of view of two related disciplines, process engineering and biotechnology, is presented together with the results of previous comparative studies. These reviews highlight possible areas where more understanding is required. This is provided in the paper by application of the flexibility index to two case studies. The flexibility index is a useful process design tool that measures the ability of the process to cope with long term changes in operation.

The Lake Tekapo experiment (LTEX): An investigation of atmospheric boundary layer processes in complex terrain

A research program on atmospheric boundary layer processes and local wind regimes in complex terrain was conducted in the vicinity of Lake Tekapo in the southern Alps of New Zealand, during two 1-month field campaigns in 1997 and 1999. The effects of the interaction of thermal and dynamic forcing were of specific interest, with a particular focus on the interaction of thermal forcing of differing scales. The rationale and objectives of the field and modeling program are described, along with the methodology used to achieve them. Specific research aims include improved knowledge of the role of surface forcing associated with varying energy balances across heterogeneous terrain, thermal influences on boundary layer and local wind development, and dynamic influences of the terrain through channeling effects. Data were collected using a network of surface meteorological and energy balance stations, radiosonde and pilot balloon soundings, tethered balloon and kite-based systems, sodar, and an instrumented light aircraft. These data are being used to investigate the energetics of surface heat fluxes, the effects of localized heating/cooling and advective processes on atmospheric boundary layer development, and dynamic channeling. A complementary program of numerical modeling includes application of the Regional Atmospheric Modeling System (RAMS) to case studies characterizing typical boundary layer structures and airflow patterns observed around Lake Tekapo. Some initial results derived from the special observation periods are used to illustrate progress made to date. In spite of the difficulties involved in obtaining good data and undertaking modeling experiments in such complex terrain, initial results show that surface thermal heterogeneity has a significant influence on local atmospheric structure and wind fields in the vicinity of the lake. This influence occurs particularly in the morning. However, dynamic channeling effects and the larger-scale thermal effect of the mountain region frequently override these more local features later in the day.

Online titrimetric and off-gas analysis for examining nitrification processes in wastewater treatment

The two steps of nitrification, namely the oxidation of ammonia to nitrite and nitrite to nitrate, often need to be considered separately in process studies. For a detailed examination, it is desirable to monitor the two-step sequence using online measurements. In this paper, the use of online titrimetric and off-gas analysis (TOGA) methods for the examination of the process is presented. Using the known reaction stoichiometry, combination of the measured signals (rates of hydrogen ion production, oxygen uptake and carbon dioxide transfer) allows the determination of the three key process rates, namely the ammonia consumption rate, the nitrite accumulation rate and the nitrate production rate. Individual reaction rates determined with the TOGA sensor under a number of operation conditions are presented. The rates calculated directly from the measured signals are compared with those obtained from offline liquid sample analysis. Statistical analysis confirms that the results from the two approaches match well. This result could not have been guaranteed using alternative online methods. As a case study, the influences of pH and dissolved oxygen (DO) on nitrite accumulation are tested using the proposed method. It is shown that nitrite accumulation decreased with increasing DO and pH. Possible reasons for these observations are discussed. (C) 2003 Elsevier Science Ltd. All rights reserved.

Dendritic branching of pyramidal cells in the visual cortex of the nocturnal owl monkey: A fractal analysis

The branching structure of neurones is thought to influence patterns of connectivity and how inputs are integrated within the arbor. Recent studies have revealed a remarkable degree of variation in the branching structure of pyramidal cells in the cerebral cortex of diurnal primates, suggesting regional specialization in neuronal function. Such specialization in pyramidal cell structure may be important for various aspects of visual function, such as object recognition and color processing. To better understand the functional role of regional variation in the pyramidal cell phenotype in visual processing, we determined the complexity of the dendritic branching pattern of pyramidal cells in visual cortex of the nocturnal New World owl monkey. We used the fractal dilation method to quantify the branching structure of pyramidal cells in the primary visual area (V1), the second visual area (V2) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively), which are often associated with color processing. We found that, as in diurnal monkeys, there was a trend for cells of increasing fractal dimension with progression through these cortical areas. The increasing complexity paralleled a trend for increasing symmetry. That we found a similar trend in both diurnal and nocturnal monkeys suggests that it was a feature of a common anthropoid ancestor.

Mid- to Late-Holocene estuarine infilling processes studied by radiocarbon dates, high resolution seismic and biofacies at Vitoria Bay, Espirito Santo, Southeastern Brazil

A Baía de Vitória é um estuário com 20 km de comprimento, morfologicamente estreito, com um regime de micromaré e, como outros estuários modernos, formado durante a última transgressão pós-glacial. A morfologia de fundo do estrato estuarino é caracterizada por um canal natural principal limitado por planícies de maré com manguezais desenvolvidos. Datações de radiocarbono originais foram obtidas para a área. Cinco idades de radiocarbono estendendo-se de 1.010 a 7.240 anos AP foram obtidas através de dois testemunhos de sedimento, representando uma sequência estratigráfica de 5 m de espessura. Os resultados indicam que até aproximadamente 4.000 anos cal. AP, as condições ambientais da Baía de Vitória eram ainda de uma baía aberta, com uma conexão livre e aberta com águas marinhas. Durante os últimos 4.000 anos a baía experimentou uma fase de regressão importante, tornando-se mais restrita em termos de circulação da água do mar e provavelmente aumentando a energia de marés. Três superfícies estratigráficas principais foram reconhecidas, limitando fácies transgressiva, transgressiva/nível de mar alto e regressiva. A morfologia do canal atual representa um diastema de maré, mostrando fácies regressivas truncadas e erodidas. Biofácies de foraminíferos, passando de ambiente marinho para ambiente salobro e de manguezais em planície de maré confirmam a interpretação sismoestratigráfica. A ausência de biofácies de mangue em um dos dois testemunhos é tambémuma indicação de ravinamento de maré atual.

Acute lead-induced vasoconstriction in the vascular beds of isolated perfused rat tails is endothelium-dependent

Chronic lead exposure induces hypertension in humans and animals, affecting endothelial function. However, studies concerning acute cardiovascular effects are lacking. We investigated the effects of acute administration of a high concentration of lead acetate (100 µΜ) on the pressor response to phenylephrine (PHE) in the tail vascular bed of male Wistar rats. Animals were anesthetized with sodium pentobarbital and heparinized. The tail artery was dissected and cannulated for drug infusion and mean perfusion pressure measurements. Endothelium and vascular smooth muscle relaxation were tested with acetylcholine (5 µg/100 µL) and sodium nitroprusside (0.1 µg/100 µL), respectively, in arteries precontracted with 0.1 µM PHE. Concentration-response curves to PHE (0.001-300 µg/100 µL) were constructed before and after perfusion for 1 h with 100 µΜ lead acetate. In the presence of endothelium (E+), lead acetate increased maximal response (Emax) (control: 364.4 ± 36, Pb2+: 480.0 ± 27 mmHg; P < 0.05) and the sensitivity (pD2; control: 1.98 ± 0.07, 2.38 ± 0.14 log mM) to PHE. In the absence of endothelium (E-) lead had no effect but increased baseline perfusion pressure (E+: 79.5 ± 2.4, E-: 118 ± 2.2 mmHg; P < 0.05). To investigate the underlying mechanisms, this protocol was repeated after treatment with 100 µM L-NAME, 10 µM indomethacin and 1 µM tempol in the presence of lead. Lead actions on Emax and pD2 were abolished in the presence of indomethacin, and partially abolished with L-NAME and tempol. Results suggest that acute lead administration affects the endothelium, releasing cyclooxygenase-derived vasoconstrictors and involving reactive oxygen species.

Animação Viral, Vanguarda Trash e o Capital Simbólico dos nossos Piores Defeitos: a Ideologia de Bruno Aleixo

"Bruno Aleixo" is a viral animation character, created by the Portuguese collective GANA, that surfaced online in 2008. Their animation works have meanwhile crossed onto the most diverse media, and have been branching out in multiple webs of narratives, constantly referring to each other, as well as constantly quoting disparate references such as film classics, chatrooms and TV ads for detergents. This paper attempts a triple analysis of this object of study: the ways in which technology has been fostering non-linear narratives while widening the available aesthetic spectrum, the ways in which processes of cultural consumerism are being reinvented in light of the web 2.0, and the use of "pseudo-nonsense" as a process of oblique cultural psychoanalysis. We will further attempt to demonstrate how new media and web networks have been contributing to a fragmentation of audiences, as well as a blurring between dominant cultures and sub-cultural phenomena; and we will end by positing that the structural principles behind the "Bruno Aleixo" series can be applied in social and cultural contexts situated at the opposite end of the spectrum of traditional expectations regarding Animation.

Electrospun silk-elastin-like fiber mats for tissue engineering applications

Protein-based polymers are present in a wide variety of organisms fulfilling structural and mechanical roles. Advances in protein engineering and recombinant DNA technology allow the design and production of recombinant protein-based polymers (rPBPs) with an absolute control of its composition. Although the application of recombinant proteins as biomaterials is still an emerging technology, the possibilities are limitless and far superior to natural or synthetic materials, as the complexity of the structural design can be fully customized. In this work, we report the electrospinning of two new genetically engineered silk-elastin-like proteins (SELPs) consisting of alternate silk- and elastin-like blocks. Electrospinning was performed with formic acid and aqueous solutions at different concentrations without addition of further agents. The size and morphology of the electrospun structures was characterized by scanning electron microscopy showing to be dependent of concentration and solvent used. Treatment with air saturated with methanol was employed to stabilize the structure and promote water insolubility through a time-dependent conversion of random coils into β-sheets (FTIR). The resultant methanol-treated electrospun mats were characterized for swelling degree (570-720%), water vapour transmission rate (1083 g/m2/day) and mechanical properties (modulus of elasticity of ~126 MPa). Furthermore, the methanol-treated SELP fiber mats showed no cytotoxicity and were able to support adhesion and proliferation of normal human skin fibroblasts. Adhesion was characterized by a filopodia-mediated mechanism. These results demonstrate that SELP fiber mats can provide promising solutions for the development of novel biomaterials suitable for tissue engineering applications.

Application to quantify fetal lung branching on rat explants

Recently, regulating mechanisms of branching morphogenesis of fetal lung rat explants have been an essential tool for molecular research. The development of accurate and reliable segmentation techniques may be essential to improve research outcomes. This work presents an image processing method to measure the perimeter and area of lung branches on fetal rat explants. The algorithm starts by reducing the noise corrupting the image with a pre-processing stage. The outcome is input to a watershed operation that automatically segments the image into primitive regions. Then, an image pixel is selected within the lung explant epithelial, allowing a region growing between neighbouring watershed regions. This growing process is controlled by a statistical distribution of each region. When compared with manual segmentation, the results show the same tendency for lung development. High similarities were harder to obtain in the last two days of culture, due to the increased number of peripheral airway buds and complexity of lung architecture. However, using semiautomatic measurements, the standard deviation was lower and the results between independent researchers were more coherent

A creative solution for rapid and inexpensive model-making in product design processes

Innovative Developments in Virtual and Physical Prototyping