Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

Characterisation of wood–water relationships and transverse anatomy and their relationship to drying degrade

Characterisation of a number of key wood properties utilising ‘state of the art’ tools was achieved for four commercial Australian hardwood species: Corymbia citriodora, Eucalyptus pilularis, Eucalyptus marginata and Eucalyptus obliqua. The wood properties were measured for input into microscopic (cellular level) and macroscopic (board level) vacuum drying models currently under development. Morphological characterisation was completed using a combination of ESEM, optical microscopy and a custom vector-based image analysis software. A clear difference in wood porosity, size, wall thickness and orientation was evident between species. Wood porosity was measured using a combination of fibre and vessel porosity. A highly sensitive microbalance and scanning laser micrometres were used to measure loss of moisture content in conjunction with directional shrinkage on micro-samples of E. obliqua to investigate the validity of measuring collapse-free shrinkage in very thin sections. Collapse-free shrinkage was characterised, and collapse propensity was verified when testing thicker samples. Desorption isotherms were calculated for each species using wood–water relations data generated from shrinkage experiments. Fibre geometry and wood shrinkage anisotropy were used to explain the observed difficulty in drying of the different species in terms of collapse and drying stress-related degrade.

Thermal decomposition of rare-earth trioxalatocobaltates

The thermal decomposition of rare-earth trioxalatocobaltates LnCo(C2O4)3 · x H2O, where Ln = La, Pr, Nd, has been studied in flowing atmospheres of air/oxygen, argon/ nitrogen, carbon dioxide and a vacuum. The compounds decompose through three major steps, viz. dehydration, decomposition of the oxalate to an intermediate carbonate, which further decomposes to yield rare-earth cobaltite as the final product. The formation of the final product is influenced by the surrounding gas atmosphere. Studies on the thermal decomposition of photodecomposed lanthanum trioxalatocobaltate and a mechanical mixture of lanthanum oxalate and cobalt oxalate in 1 : 2 molar ratio reveal that the decomposition behaviour of the two samples is different. The drawbacks of the decomposition scheme proposed earlier have been pointed out, and logical schemes based on results obtained by TG, DTA, DTG, supplemented by various physico-chemical techniques such as gas and chemical analyses, IR and mass spectroscopy, surface area and magnetic susceptibility measurements and X-ray powder diffraction methods, have been proposed for the decomposition in air of rare-earth trioxalatocobaltates as well as for the photoreduced lanthanum salt and a mechanical mixture of lanthanum and cobalt oxalates.

Degenerate surface modes in a magnetized plasma

Electromagnetic surface waves propagating along the plasma-vacuum interface parallel to an applied magnetic field are studied. New modes for which the field components are degenerate, not reported in the earlier investigation of Kotsarenko and Fedorchenko (1969), are found and discussed. These modes, which propagate up to the plasma frequency for all values of the magnetic field, start as forward waves at low frequency but smoothly change into the backward mode as the frequency increases.

Incomplete decomposition of alkali metal azides

The thermal decomposition of sodium azide has been studied in the temperature range 240–360°C in vacuum and under pressure of an inert gas, argon. The results show that the decomposition is partial 360°C. From the observations made in the present work, namely: (i) the decomposition is incomplete both under vacuum and inert gas; (ii) mass spectrometric studies do not reveal any decrease in the intensity of the background species, CO+2, CO+, H2O+, and (iii) sodium metal remains in the ‘free state’ as seen by the formation of a metallic mirror at temperatures above 300°C, it has been argued that the partial nature of decompostion is due to the confinement of the decomposition to intermosaic regions within the lattice.

Thermal decomposition of titanyl oxalates II : Kinetics of decomposition of barium titanyl oxalate

Kinetics of the thermal decomposition of barium titanyl oxalate have been studied. Decomposition of the anhydrous oxalate is complex and deceleratory throughout. Kinetics of decomposition of the intermediate carbonate Ba2Ti2O5CO3 is greatly influenced by the thermal effects during its formation. The sigmoidal (α, t) curves obey a power law equation followed by first order decay. Presence of carbon in the vacuum prepared carbonate has a strong deactivating effect. Decomposition of the carbonate is accompanied by growth in particle size of the product, barium titanate.

Impulse Voltage Breakdown Characteristics of Point-Plane Gaps at Low Pressures

Practical applications of vacuum as an insulator necessitated determining the low-pressure breakdown characteristics of long gap lengths of a point-plane electrode system. The breakdown voltage has been found to vary as the square root of the gap length. Further, with the point electrode as the anode, the values of the breakdown voltages obtained have been found to be larger than those obtained with a plane-parallel electrode system at a corresponding gap length. By applying the theory of the anode heating mechanism as the cause for breakdown, the results have been justified, and by utilizing a field efficiency factor which is the ratio of the average to maximum field, an empirical criterion has been developed. This criterion helps in calculating the breakdown voltage of a nonuniform gap system by the knowledge of the breakdown voltage of a plane-parallel electrode system.

Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators

Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression. Polypurine/polypyrimidine sequences [poly(Pu/Py)] have been observed in the vicinity of promoters and within the transcribed regions of many genes. To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding β-galactosidase), and studied its expression in vivo. We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells. On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells. Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V. Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs. On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators.

Effect of oxygen partial pressure on the growth of zinc micro and nanostructures

Zinc micro and nanostructures were synthesized in vacuum by condensing evaporated zinc on Si substrate at different gas pressures. The morphology of the grown Zn structures was found to be dependent on the oxygen partial pressure. Depending on oxygen partial pressure it varied from two-dimensional microdisks to one-dimensional nanowire. The morphology and structural properties of the grown micro and nanostructures were studied by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Transmission electron microscopy (TEM) studies on the grown Zn nanowires have shown that they exhibit core/shell-like structures, where a thin ZnO layer forms the shell. A possible growth mechanism behind the formation of different micro and nanostructures has been proposed. In addition, we have synthesized ZnO nanocanal-like structures by annealing Zn nanowires in vacuum at 350 °C for 30 min.

Influence of Solvents on the MWCNT/Adhesive Grade Epoxy Nanocomposites Preparation

In this article, the effect of two solvents, namely dimethyl formamide (DMF) and N-methyl pyrrolidone (NMP), on the dispersion effectiveness and the resulting electrical and mechanical properties of multi-walled carbonanotubes (MNCNT) filled structural adhesive grade epoxy nanocomposites was studied. The solvents were used mainly to reduce the viscosity of the resin system to effectively disperse the nanofiller. The dispersion was carried out under vacuum using high energy sonic waves. SEM was undertaken to study the dispersion effectiveness. Electrical resistivity, tensile properties, and glass transition of the nanocomposites were studied. Between DMF and NMP, the former proved better in terms of dispersion effectiveness and the resulting electrical and mechanical properties of the nanocomposites. Addition of MWCNT into AV138M resulted in an increase in glass transition temperature irrespective of the solvent used and in both cases percolation threshold was found with respect to reduction in electrical resistivity of the nanocomposites. Less agglomeration and hence better interaction between CNT and epoxy was observed in the samples prepared using DMF compared with that using NMP.

The effect of lignin content and lignin modification on Norway spruce wood properties and decay resistance

Three different Norway spruce cutting clones growing in three environments with different soil and climatic conditions were studied. The purpose was to follow variation in the radial growth rate, wood properties and lignin content and to modify wood lignin with a natural monolignol, coniferyl alcohol, by making use of inherent wood peroxidases. In addition, the incorporation of chlorinated anilines into lignin was studied with synthetic model compounds and synthetic lignin preparations to show whether unnatural compounds originating from pesticides could be bound in the lignin polymer. The lignin content of heartwood, sapwood and earlywood was determined by applying Fourier transform infrared (FTIR) spectroscopy and a principal component regression (PCR) technique. Wood blocks were treated with coniferyl alcohol by using a vacuum impregnation method. The effect of impregnation was assessed by FTIR and by a fungal decay test. Trees from a fertile site showed the highest growth rate and sapwood lignin content and the lowest latewood proportion, weight density and modulus of rupture (MOR). Trees from a medium fertile site had the lowest growth rate and the highest latewood proportion, weight density, modulus of elasticity (MOE) and MOR. The most rapidly growing clone showed the lowest latewood proportion, weight density, MOE and MOR. The slowest growing clone had the lowest sapwood lignin content and the highest latewood proportion, weight density, MOE and MOR. Differences between the sites and clones were small, while fairly large variation was found between the individual trees and growing seasons. The cutting clones maintained clone-dependent wood properties in the different growing sites although variation between trees was high and climatic factors affected growth. The coniferyl alcohol impregnation increased the content of different lignin-type phenolic compounds in the wood as well as wood decay resistance against a white-rot fungus, Coriolus versicolor. During the synthetic lignin preparation 3,4-dichloroaniline became bound by a benzylamine bond to β-O-4 structures in the polymer and it could not be released by mild acid hydrolysis. The natural monolignol, coniferyl alcohol, and chlorinated anilines could be incorporated into the lignin polymer in vivo and in vitro, respectively.

Highly stable oscillator for quartz crystal thickness monitor

A highly stable oscillator used in a quartz crystal thickness monitor for monitoring the rate of evaporation and total thickness of film during thin film deposition is reported. The design aspects of the oscillator and its long term stability, which enhances the reproducibility and the performance of the thickness monitor, are discussed. The stability of the oscillator at defined conditions is tested and compared with the conventional transistorized oscillator and the IC oscillator using inverters. The oscillator is coupled to the crystal monitor and its performance is studied in an evaporation system by evaporating different materials

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

Synthesis of liquid crystalline benzothiazole base derivatives: A study of their optical and electrical properties

Two new donor-acceptor type liquid crystalline semiconductors based on benzothiazole have been synthesized. Their structural, photophysical and electronic properties were investigated using X-ray diffraction, atomic force microscopy, cyclic voltammetry, UV-Vis, photoluminescence, and Raman spectroscopy. The liquid crystalline behaviour of the molecules was thoroughly examined by differential scanning calorimetry (DSC) and optical polarizing microscope. The DSC and thermogravimetric analysis (TGA) show that these materials posses excellent thermal stability and have decomposition temperatures in excess of 300 degrees C. Beyond 160 degrees C both molecules show a smectic A liquid crystalline phase that exists till about 240 degrees C. Field-effect transistors were fabricated by vacuum evaporating the semiconductor layer using standard bottom gate/top contact geometry. The devices exhibit p-channel behaviour with hole mobilities of 10(-2) cm(2)/Vs. (C) 2009 Elsevier B.V. All rights reserved.