Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

Comparison between pre-enrichment in single- or double-strength buffered peptone water for recovery of Salmonella enterica serovar Typhimurium DT104 from acidic marinade sauces containing spices.

The ability of the standard pre-enrichment procedure in buffered peptone water (BPW) to recover Salmonella Typhimurium from acidic marinade sauces containing spices was tested by inoculating marinade sauces with known numbers of an antibiotic-resistant marker strain of Salmonella Typhimurium DT104 prior to pre-enrichment. Viable numbers of salmonellae present in BPW after 24h incubation depended on the inoculum level. If initial cell numbers were low (below 103 cfu per 250 ml BPW) final cell concentrations were also low and, in some cases, no growth occurred. The problem was overcome by use of double-strength BPW that neutralised the acidity and allowed good recovery from otherwise inhibitory marinade sauces.

Nop53p interacts with 5.8S rRNA co-transcriptionally, and regulates processing of pre-rRNA by the exosome

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Delta nop53/GAL:NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.

Fabrics of pre- and syntectonic granite plutons and chronology of shear zones in the Eastern Borborema Province, NE Brazil

We used the fabrics of two granite plutons and U/Pb (SHRIMP) zircon ages to constrain the tectonic evolution of the E-trending Patos shear zone (Borborema Province, NE Brazil). The pre-tectonic Teixeira batholith consists of an amphibole leucogranite locally with aegirine-augite. Zircons from a syenogranite yielded crystallization ages of 591 +/- 5 Ma. The batholith fabrics were determined by anisotropy of magnetic susceptibility (AMS) and mineral shape preferred orientation. The fabrics support pre-transcurrent batholith emplacement, as evidenced by: (i) magmatic/magnetic fabrics in low susceptibility (<0.35 mSI) leucogranites highly discordant to the regional host rock structure, and (ii) concordant magnetic fabrics restricted to high susceptibility (>1 mSI) corridors connected to shear zones branching off from Patos. One of these satellite shear zones controlled the syntectonic emplacement of the Serra Redonda pluton, which yields a crystallization age of 576 +/- 3 Ma. This late shearing event marks the peak regional deformation that, south of Patos, was coupled to crustal shortening nearly perpendicular to the shear belt. The chronology of the deformational events indicates that the major shear zones of the eastern Borborema are late structures active after the crustal blocks amalgamated. (C) 2007 Elsevier Ltd. All rights reserved.

Efeito da ciclagem mecânica na deflexão de cúspides de pré-molares restaurados pela técnica direta e indireta: estudo com strain gauge

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Study of the components of quality in SO2-free wines obtained by innovative vinification protocols. Evaluation of the pre-fermentative addition of lysozyme and oenological tannins.

The research performed during the PhD candidature was intended to evaluate the quality of white wines, as a function of the reduction in SO2 use during the first steps of the winemaking process. In order to investigate the mechanism and intensity of interactions occurring between lysozyme and the principal macro-components of musts and wines, a series of experiments on model wine solutions were undertaken, focusing attention on the polyphenols, SO2, oenological tannins, pectines, ethanol, and sugar components. In the second part of this research program, a series of conventional sulphite added vinifications were compared to vinifications in which sulphur dioxide was replaced by lysozyme and consequently define potential winemaking protocols suitable for the production of SO2-free wines. To reach the final goal, the technological performance of two selected yeast strains with a low aptitude to produce SO2 during fermentation were also evaluated. The data obtained suggested that the addition of lysozyme and oenological tannins during the alcoholic fermentation could represent a promising alternative to the use of sulphur dioxide and a reliable starting point for the production of SO2-free wines. The different vinification protocols studied influenced the composition of the volatile profile in wines at the end of the alcoholic fermentation, especially with regards to alcohols and ethyl esters also a consequence of the yeast’s response to the presence or absence of sulphites during fermentation, contributing in different ways to the sensory profiles of wines. In fact, the aminoacids analysis showed that lysozyme can affect the consumption of nitrogen as a function of the yeast strain used in fermentation. During the bottle storage, the evolution of volatile compounds is affected by the presence of SO2 and oenological tannins, confirming their positive role in scaveging oxygen and maintaining the amounts of esters over certain levels, avoiding a decline in the wine’s quality. Even though a natural decrease was found on phenolic profiles due to oxidation effects caused by the presence of oxygen dissolved in the medium during the storage period, the presence of SO2 together with tannins contrasted the decay of phenolic content at the end of the fermentation. Tannins also showed a central role in preserving the polyphenolic profile of wines during the storage period, confirming their antioxidant property, acting as reductants. Our study focused on the fundamental chemistry relevant to the oxidative phenolic spoilage of white wines has demonstrated the suitability of glutathione to inhibit the production of yellow xanthylium cation pigments generated from flavanols and glyoxylic acid at the concentration that it typically exists in wine. The ability of glutathione to bind glyoxylic acid rather than acetaldehyde may enable glutathione to be used as a ‘switch’ for glyoxylic acid-induced polymerisation mechanisms, as opposed to the equivalent acetaldehyde polymerisation, in processes such as microoxidation. Further research is required to assess the ability of glutathione to prevent xanthylium cation production during the in-situ production of glyoxylic acid and in the presence of sulphur dioxide.

Aerobic biodegradation of chlorinated solvents and plastics in batch and continuous-flow bioreactors: process development, and identification of suitable chemical pre-Treatments

The purpose of the first part of the research activity was to develop an aerobic cometabolic process in packed bed reactors (PBR) to treat real groundwater contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). In an initial screening conducted in batch bioreactors, different groundwater samples from 5 wells of the contaminated site were fed with 5 growth substrates. The work led to the selection of butane as the best growth substrate, and to the development and characterization from the site’s indigenous biomass of a suspended-cell consortium capable to degrade TCE with a 90 % mineralization of the organic chlorine. A kinetic study conducted in batch and continuous flow PBRs and led to the identification of the best carrier. A kinetic study of butane and TCE biodegradation indicated that the attached-cell consortium is characterized by a lower TCE specific degredation rates and by a lower level of mutual butane-TCE inhibition. A 31 L bioreactor was designed and set up for upscaling the experiment. The second part of the research focused on the biodegradation of 4 polymers, with and with-out chemical pre-treatments: linear low density polyethylene (LLDPE), polyethylene (PP), polystyrene (PS) and polyvinyl chloride (PVC). Initially, the 4 polymers were subjected to different chemical pre-treatments: ozonation and UV/ozonation, in gaseous and aqueous phase. It was found that, for LLDPE and PP, the coupling UV and ozone in gas phase is the most effective way to oxidize the polymers and to generate carbonyl groups on the polymer surface. In further tests, the effect of chemical pretreatment on polyner biodegrability was studied. Gas-phase ozonated and virgin polymers were incubated aerobically with: (a) a pure strain, (b) a mixed culture of bacteria; and (c) a fungal culture, together with saccharose as a co-substrate.

Cell shape-dependent early responses of fibroblasts to cyclic strain

Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000μm(2)) were micro-contact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size.

Interpretation of fluid inclusions in quartz deformed by weak ductile shearing: reconstruction of differential stress magnitudes and pre-deformation fluid properties

A well developed theoretical framework is available in which paleofluid properties, such as chemical composition and density, can be reconstructed from fluid inclusions in minerals that have undergone no ductile deformation. The present study extends this framework to encompass fluid inclusions hosted by quartz that has undergone weak ductile deformation following fluid entrapment. Recent experiments have shown that such deformation causes inclusions to become dismembered into clusters of irregularly shaped relict inclusions surrounded by planar arrays of tiny, new-formed (neonate) inclusions. Comparison of the experimental samples with a naturally sheared quartz vein from Grimsel Pass, Aar Massif, Central Alps, Switzerland, reveals striking similarities. This strong concordance justifies applying the experimentally derived rules of fluid inclusion behaviour to nature. Thus, planar arrays of dismembered inclusions defining cleavage planes in quartz may be taken as diagnostic of small amounts of intracrystalline strain. Deformed inclusions preserve their pre-deformation concentration ratios of gases to electrolytes, but their H2O contents typically have changed. Morphologically intact inclusions, in contrast, preserve the pre-deformation composition and density of their originally trapped fluid. The orientation of the maximum principal compressive stress (σ1σ1) at the time of shear deformation can be derived from the pole to the cleavage plane within which the dismembered inclusions are aligned. Finally, the density of neonate inclusions is commensurate with the pressure value of σ1σ1 at the temperature and time of deformation. This last rule offers a means to estimate magnitudes of shear stresses from fluid inclusion studies. Application of this new paleopiezometer approach to the Grimsel vein yields a differential stress (σ1–σ3σ1–σ3) of ∼300 MPa∼300 MPa at View the MathML source390±30°C during late Miocene NNW–SSE orogenic shortening and regional uplift of the Aar Massif. This differential stress resulted in strain-hardening of the quartz at very low total strain (<5%<5%) while nearby shear zones were accommodating significant displacements. Further implementation of these experimentally derived rules should provide new insight into processes of fluid–rock interaction in the ductile regime within the Earth's crust.

Does prior receipt of a rifamycin antibiotic increase the risk of acquiring an infection due to a rifamycin-resistant strain of Clostridium difficile?

Objectives. To examine the association between prior rifamycin exposure and later development of C. difficile infection (CDI) caused by a rifamycin-resistant strain of C. difficile , and to compare patient characteristics between rifamycin-resistant strains of C. difficile infection and rifamycin-susceptible strains of C. difficile infection. ^ Methods. A case-control study was performed in a large university-affiliated hospital in Houston, Texas. Study subjects were patients with C. difficile infection acquired at the hospital with culture-positive isolates of C. difficile with which in vitro rifaximin and rifampin susceptibility has been tested. Prior use of rifamycin, demographic and clinical characteristics was compared between case and control groups using univariate statistics. ^ Results. A total of 49 C. difficile strains met the study inclusion criteria for rifamycin-resistant case isolates, and a total of 98 rifamycin-susceptible C. difficile strains were matched to case isolates. Of 49 case isolates, 12 (4%) were resistant to rifampin alone, 12 (4%) were resistant to rifaximin alone, and 25 (9%) were resistant to both rifampin and rifaximin. There was no significant association between prior rifamycin use and rifamycin-resistant CDI. Cases and controls did not differ according to demographic characteristics, length of hospital stay, known risk factors of CDI, type of CDI-onset, and pre-infection medical co-morbidities. Our results on 37 rifaximin-resistant isolates (MIC ≥32 &mgr;g/ml) showed more than half of isolates had a rifaximin MIC ≥256 &mgr;g/ml, and out of these isolates, 19 isolates had MICs ≥1024 &mgr;g/ml. ^ Conclusions. Using a large series of rifamycin-non-susceptible isolates, no patient characteristics were independently associated with rifamycin-resistant CDI. This data suggests that factors beyond previous use of rifamycin antibiotics are primary risk factors for rifamycin-resistant C. difficile. ^

Representative analyses of pre-tectonic metabasic rocks and metapelitic gneiss from Sverdrupfjella, East Antarctica

Extensive high-grade polydeformed metamorphic provinces surrounding Archaean cratonic nuclei in the East Antarctic Shield record two tectono-thermal episodes in late Mesoproterozoic and late Neoproterozoic-Cambrian times. In Western Dronning Maud Land, the high-grade Mesoproterozoic Maud Belt is juxtaposed against the Archaean Grunehogna Province and has traditionally been interpreted as a Grenvillian mobile belt that was thermally overprinted during the Early Palaeozoic. Integration of new U-Pb sensitive high-resolution ion microprobe and conventional single zircon and monazite age data, and Ar-Ar data on hornblende and biotite, with thermobarometric calculations on rocks from the H.U. Sverdrupfjella, northern Maud Belt, resulted in a more complex P-T-t evolution than previously assumed. A c. 540?Ma monazite, hosted by an upper ampibolite-facies mineral assemblage defining a regionally dominant top-to-NW shear fabric, provides strong evidence for the penetrative deformation in the area being of Pan-African age and not of Grenvillian age as previously reported. Relics of an eclogite-facies garnet-omphacite assemblage within strain-protected mafic boudins indicate that the peak metamorphic conditions recorded by most rocks in the area (T = 687-758°C, P = 9·4-11·3?kbar) were attained subsequent to decompression from P > 12·9?kbar. By analogy with limited U-Pb single zircon age data and on circumstantial textural grounds, this earlier eclogite-facies metamorphism is ascribed to subduction and accretion around 565?Ma. Post-peak metamorphic K-metasomatism under amphibolite-facies conditions is ascribed to the intrusion of post-orogenic granite at c. 480?Ma. The recognition of extensive Pan-African tectonism in the Maud Belt casts doubts on previous Rodinia reconstructions, in which this belt takes a pivotal position between East Antarctica, the Kalahari Craton and Laurentia. Evidence of late Mesoproterozoic high-grade metamorphism during the formation of the Maud Belt exists in the form of c. 1035?Ma zircon overgrowths that are probably related to relics of granulite-facies metamorphism recorded from other parts of the Maud Belt. The polymetamorphic rocks are largely derived from a c. 1140?Ma volcanic arc and 1072 ± 10?Ma granite.

Deformation of sand in plane strain and oxisymmetric compression

Small scale laboratory experiments, in which the specimen is considered to represent an element of soil in the soil mass, are essential to the evolution of fundamental theories of mechanical behaviour. In this thesis, plane strain and axisymmetric compression tests, performed on a fine sand, are reported and the results are compared with various theoretical predictions. A new apparatus is described in which cuboidal samples can be tested in either axisymmetric compression or plane strain. The plane strain condition is simulated either by rigid side platens, in the conventional manner, or by flexible side platens which also measure the intermediate principal stress. Close control of the initial porosity of the specimens is achieved by a vibratory method of sample preparation. The strength of sand is higher in plane strain than in axisymmetric compression, and the strains required to mobilize peak strength are much smaller. The difference between plane strain and axisymmetric compression behaviour is attributed to the restrictions on particle movement enforced by the plane strain condition; this results in an increase in the frictional component of shear strength. The stress conditions at failure in plane strain, including the intermediate principal stress, are accurately predicted by a theory based on the stress- dilatancy interpretation of Mohr's circles. Detailed observations of rupture modes are presented and measured rupture plane inclinations are predicted by the stress-dilatancy theory. Although good correlation with the stress-dilatancy theory is obtained during virgin loading, in both axisymmetric compression and plane strain, the stress-dilatancy rule is only obeyed during reloading if the specimen has been unloaded to approximate ambient stress conditions. The shape of the stress-strain curves during pre-peak deformation, in both plane strain and axisymmetric compression, is accurately described bv a combined parabolic-hyperbolic specification.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.