New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Alternative nighttime nutrition regimens in glycogen storage disease type I: a controlled crossover study

BACKGROUND Traditional approaches for nighttime glycemic control in glycogen storage disease type I (GSDI) include continuous tube feeding, or ingestion of uncooked corn starch (CS) at bedtime. A modified corn starch (MCS) has been shown to prolong euglycemia in some patients. The aim of this study was to evaluate whether stable nighttime glucose control can be achieved with other types of slowly digested carbohydrates in adult GSDI patients. METHODS In this cross-over study, nocturnal glucose control and fasting times were assessed with three different nocturnal nutrition regimens in five patients, using continuous glucose monitoring (CGMS) in an outpatient everyday life setting. For each patient, continuous glucose profiles were measured after ingestion of (1) CS, (2) MCS or (3) a pasta meal at bedtime, during 5 to 6 consecutive nights for each regimen. RESULTS Stable nocturnal glucose control was achieved for all patients with a pasta meal, with a mean duration of glycemia >3.5 mmol/l of 7.6 h (range 5.7-10.8), and >4 mmol/l of 7 h (5.2-9.2), similar to CS and MCS. Fasting glucose before breakfast on workdays (after 7.1 ± 0.8 h) was not significantly different between the three interventions (CS 4.1 ± 0.5 mmol/l, MCS 4.6 ± 0.7 mmol/l, pasta 4.3 ± 0.9 mmol/l). During prolonged fasting on weekends, longer duration of normoglycemia was achieved with CS or MCS than with pasta. CONCLUSION Consumption of cooked pasta is a suitable and more palatable alternative to uncooked corn starch to achieve nighttime glucose control in adult patients with GSDI.

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

Physical properties of ODP Leg 178 sites

We have reanalyzed the porosity, bulk density, and seismic velocity information collected from continental rise Sites 1095, 1096, and 1101 during the drilling of Ocean Drilling Program (ODP) Leg 178 (Fig. F1). The purpose is to provide a comprehensive composite digital set of data readily available for future studies aimed at well-seismic correlation. The work originates from the occurrence of overlapping sets of physical parameters and acoustic velocity collected by different methods (downhole logging, core logging, laboratory determination, and derivation from seismic data) and from different holes at the same site. These data do not always provide the same information because of difficulties encountered at each specific hole or methodological differences. In addition, a basic correlation between these parameters and onsite multichannel seismic (MCS) data is presented.

Ages and two-way travel times of ODP Sites 180-1109, 180-1115 and 180-1118

Synthetic seismograms are constructed from check shot-corrected velocity and density measurements collected during Ocean Drilling Program (ODP) Leg 180 at Sites 1109, 1115, and 1118. The synthetic seismograms facilitate direct correlation of a coincident multichannel seismic (MCS) profile with borehole data collected at the three sites. The MCS data and the synthetic seismograms correlate very well, with most major reflectors successfully reproduced in the synthetics. Our results enable a direct calibration of the MCS data in terms of age, paleoenvironment, and subsidence history. Seismic reflectors are time correlative within stratigraphic resolution but are often observed to result from different lithologies across strike. Our results facilitate the extrapolation of the sedimentation history into an unsampled section of Site 1118 and enable a full correlation between the three sites using all the data collected during ODP Leg 180. This study forms the foundation for regionalizing the site data to the northern margin of the Woodlark Basin, where the transition from continental rifting to seafloor spreading is taking place.

Biogenic silica and silicic acid benthic fluxes of a North-eastern Atlantic Abyssal Locality

Within the framework of the EU-funded BENGAL programme, the effects of seasonality on biogenic silica early diagenesis have been studied at the Porcupine Abyssal Plain (PAP), an abyssal locality located in the northeast Atlantic Ocean. Nine cruises were carried out between August 1996 and August 1998. Silicic acid (DSi) increased downward from 46.2 to 213 µM (mean of 27 profiles). Biogenic silica (BSi) decreased from ca. 2% near the sediment-water interface to <1% at depth. Benthic silicic acid fluxes as measured from benthic chambers were close to those estimated from non-linear DSi porewater gradients. Some 90% of the dissolution occurred within the top 5.5 cm of the sediment column, rather than at the sediment-water interface and the annual DSi efflux was close to 0.057 mol Si/m**2/yr. Biogenic silica accumulation was close to 0.008 mol Si/m**2/yr and the annual opal delivery reconstructed from sedimentary fluxes, assuming steady state, was 0.065 mol Si/m**2/yr. This is in good agreement with the mean annual opal flux determined from sediment trap samples, averaged over the last decade (0.062 mol Si/m**2/yr). Thus ca. 12% of the opal flux delivered to the seafloor get preserved in the sediments. A simple comparison between the sedimentation rate and the dissolution rate in the uppermost 5.5 cm of the sediment column suggests that there should be no accumulation of opal in PAP sediments. However, by combining the BENGAL high sampling frequency with our experimental results on BSi dissolution, we conclude that non-steady state processes associated with the seasonal deposition of fresh biogenic particles may well play a fundamental role in the preservation of BSi in these sediments. This comes about though the way seasonal variability affects the quality of the biogenic matter reaching the seafloor. Hence it influences the intrinsic dissolution properties of the opal at the seafloor and also the part played by non-local mixing events by ensuring the rapid transport of BSi particles deep into the sediment to where saturation is reached.

Seismic Data from MSM21/4 to Knipovich Ridge on the western Svalbard margin

The seismic data were acquired north of the Knipovich Ridge on the western Svalbard margin during cruise MSM21/4. They were recorded using a Geometrics GeoEel streamer of either 120 channels (profiles p100-p208) or 88 channels (profiles p300-p805) with a group spacing of 1.56 m and a sampling rate of 2 kHz. A GI-Gun (2×1.7 l) with a main frequency of ~150 Hz was used as a source and operated at a shot interval of 6-8 s. Processing of profiles p100-p208 and p600-p805: Positions for each channel were calculated by backtracking along the profiles from the GI-Gun GPS positions. The shot gathers were analyzed for abnormal amplitudes below the seafloor reflection by comparing neighboring traces in different frequency bands within sliding time windows. To suppress surface-generated water noise, a tau-p filter was applied in the shot gather domain. Common mid-point (CMP) profiles were then generated through crooked-line binning with a CMP spacing of 1.5625 m. A zero-phase band-pass filter with corner frequencies of 60 Hz and 360 Hz was applied to the data. Based on regional velocity information from MCS data [Sarkar, 2012], an interpolated and extrapolated 3D interval velocity model was created below the digitized seafloor reflection of the high-resolution streamer data. This velocity model was used to apply a CMP stack and an amplitude-preserving Kirchhoff post-stack time migration. Processing of profiles p400-p500: Data were sampled at 0.5 ms and sorted into common midpoint (CMP) domain with a bin spacing of 5 m. Normal move out correction was carried out with a velocity of 1500 m s-1 and an Ormsby bandpass filter with corner frequencies at 40, 80, 600 and 1000 Hz was applied. The data were time migrated using the water velocity.

Using modular neural networks to model self-consciousness and self-representation for artificial entities

Self-consciousness implies not only self or group recognition, but also real knowledge of one’s own identity. Self-consciousness is only possible if an individual is intelligent enough to formulate an abstract self-representation. Moreover, it necessarily entails the capability of referencing and using this elf-representation in connection with other cognitive features, such as inference, and the anticipation of the consequences of both one’s own and other individuals’ acts. In this paper, a cognitive architecture for self-consciousness is proposed. This cognitive architecture includes several modules: abstraction, self-representation, other individuals'representation, decision and action modules. It includes a learning process of self-representation by direct (self-experience based) and observational learning (based on the observation of other individuals). For model implementation a new approach is taken using Modular Artificial Neural Networks (MANN). For model testing, a virtual environment has been implemented. This virtual environment can be described as a holonic system or holarchy, meaning that it is composed of autonomous entities that behave both as a whole and as part of a greater whole. The system is composed of a certain number of holons interacting. These holons are equipped with cognitive features, such as sensory perception, and a simplified model of personality and self-representation. We explain holons’ cognitive architecture that enables dynamic self-representation. We analyse the effect of holon interaction, focusing on the evolution of the holon’s abstract self-representation. Finally, the results are explained and analysed and conclusions drawn.

Methodological guidelines for reusing general ontologies

Currently, there is a great deal of well-founded explicit knowledge formalizing general notions, such as time concepts and the part_of relation. Yet, it is often the case that instead of reusing ontologies that implement such notions (the so-called general ontologies), engineers create procedural programs that implicitly implement this knowledge. They do not save time and code by reusing explicit knowledge, and devote effort to solve problems that other people have already adequately solved. Consequently, we have developed a methodology that helps engineers to: (a) identify the type of general ontology to be reused; (b) find out which axioms and definitions should be reused; (c) make a decision, using formal concept analysis, on what general ontology is going to be reused; and (d) adapt and integrate the selected general ontology in the domain ontology to be developed. To illustrate our approach we have employed use-cases. For each use case, we provide a set of heuristics with examples. Each of these heuristics has been tested in either OWL or Prolog. Our methodology has been applied to develop a pharmaceutical product ontology. Additionally, we have carried out a controlled experiment with graduated students doing a MCs in Artificial Intelligence. This experiment has yielded some interesting findings concerning what kind of features the future extensions of the methodology should have.

Variation throughout the tree stem in the physical-mechanical properties of the wood of Abies alba Mill. from the Spanish Pyrenees

This study analyses the variation of main physical-mechanical properties of wood along the longitudinal and radial directions of the tree for Abies alba Mill. growing in the Spanish Pyrenees. Small clear specimens were used to study the properties of volumetric shrinkage (VS), density (?), hardness (H), bending strength (MOR), modulus of elasticity (MOE), maximum compressive strength parallel to the grain (MCS) and impact strength (K). Several models of properties variation in the longitudinal and radial directions were analyzed. Main trends of variation of properties throughout the tree stem were identified although none of them could be fitted to predictive statistical models. Along the longitudinal direction, the properties studied followed a downward trend from the base to the crown, which was not significant in all cases, indicating that no differences in quality existed. Throughout the radial direction the trend is upward for the first 40-50 growth rings, after which it slopes downwards, more gently at first until rings 70-75 and then more steeply. This behaviour is related to variation in wood structure from the pith to the bark, depending on whether the wood is juvenile, sapwood or heartwood, and to wood maturity and microfibril angle. Authors encourage carrying further studies on other populations of A. alba in the Spanish Pyrenees to check if the trends found in this study apply to other provenances.

Caracterización físico-mecánica de la madera de Abies alba Miller procedente del pirineo español mediante probetas libres de defectos

El objetivo de esta investigación es proporcionar valores actualizados y representativos de las propiedades físicas y mecánicas de la madera de abeto del Pirineo español para permitir su comparación con otras procedencias y especies maderables. Los ensayos se realizaron a partir de probetas libres de defectos obtenidas a lo largo del fuste y se determinaron las propiedades de acuerdo a las normas UNE de los ensayos correspondientes. Se concluye que la madera es ligera (ρ = 0,48 g/cm3), blanda (H = 1,71 mm-1) y moderadamente nerviosa (v = 39,43%). La resistencia a flexión (MOR = 78,70 N/mm2) es baja y a compresión (MCS =  44,88 N/mm2) media. Presenta un comportamiento al impacto medio (K = 41,46 N/mm) y bajo a hienda (C = 19,92 N/mm), tracción perpendicular tangencial y radial (1,71 y 1,68 N/mm2). Las diferencias obtenidas con respecto a otras procedencias de esta especie pueden deberse a que el abeto se encuentra en su límite de distribución geográfica y en consecuencia las condiciones específcas de sitio tienen mayor influencia en las propiedades de la madera.

HIP HOP COMO PROCESSO COMUNICACIONAL E SOCIABILIDADE PARA JOVENS INDÍGENAS DE DOURADOS – MS

Estudo sobre o Hip Hop como processo comunicacional e sociabilidade entre jovens indígenas de Dourados, Mato Grosso do Sul, para verificar quais os principais objetivos da prática do movimento Hip Hop, compreender se serve como comunicação, contribui para o fortalecimento da língua guarani ou gera novas tensões sociais na reserva. Para tanto, foi analisado aspectos históricos do movimento, passando pelos Estudos Culturais, e como Movimento Social, dando início à discussão de uma voz alternativa por meio do Hip Hop. Do ponto de vista metodológico, trata-se de um de estudo de caso, com representantes dos grupos de jovens Brô Mc's e Jovens Conscientes, das reservas Jaguapirú e Bororó, das etnias Guarani-Kaiowá de Dourados (MS). Foram realizadas entrevistas semiestruturadas junto a jovens que participaram das oficinas de hip hop, das lideranças indígenas e professores. A investigação é complementada pela pesquisa bibliográfica, documental e análises das letras de rap em confrontação com as visões da imprensa, a partir da análise dos jornais Diário MS e O Progresso. Os resultados apontam que os jovens se apropriam de uma cultura global para transformar o ambiente local com objetivo de preservar a língua guarani, uma alternativa para o conhecimento, logo para não seguirem caminhos como o das drogas. Negociando falas sobre sua realidade, dentro e fora da reserva, já que nos meios de comunicação locais há pouco espaço para a voz dos indígenas e dentro da reserva ainda há contestação do movimento em um contexto político, na tentativa de atingir uma cultura “pura”, devido à preocupação dos mais velhos com a perda de território.

Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells

The cysteinyl leukotrienes (cys-LTs) LTC4, LTD4, and LTE4 are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC4, LTD4, and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC4, but only partially to LTD4. Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC4 and UDP (≈3 log10-fold shifts in dose-response for each agonist) over LTD4 (1.3 log10-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.