In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.     

Suitability of three Ostrinia species as hosts for Macrocentrus cingulum： A comparison of their encapsulation abilities

Two cornborer species, Ostrinia furnacalis （Lepidoptera： Crambidae） and O. nubilalis, are major corn pests in Asia and Europe, respectively. In both continents, the larval endoparasitoid Macrocentrus cingulum （Hymenoptera： Braconidae） develops on another, closely related stemborer, O. scapulalis, which feeds on mugwort and other dicotyledons. M. cingulum also emerges from O. furnacalis in Asia and （9. nubilalis in North America, but not from O. nubilalis in Europe. We assessed the ability of three populations of each of the three Ostrinia species to encapsulate foreign bodies of a size similar to that of a M. cingulum egg. We conclude that variations in encapsulation ability alone cannot account for the differences observed in the field between parasite emergence rates in these different host species and geographic areas.     

Relationship of winter concealment habitat quality on pool use by juvenile spring Chinook salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) in the Grande Ronde River Basin,Oregon USA

Winter concealment habitat quality was assessed and its use by juvenile spring Chinook salmon (Oncorhynchus tshawytscha) quantified in three hatching areas of the Grande Ronde River Basin, Oregon USA. Fish densities were significantly higher in pools with a higher winter concealment habitat index than pools with a lower index. The mean fork length and mean growth rate of fish did not differ between pools with a higher or lower winter concealment habitat index, even though residual fish were significantly larger than fish that emigrated. Biomass–density was significantly higher in pools with a higher winter concealment habitat index than pools with a lower index in all three hatching areas. Biomass–density was positively associated with the amount of cobble substrate (10–24.9 cm/m²) in all three hatching areas, and inversely associated with embeddedness in two of the hatching areas. Results of this study indicate that enhancing winter concealment habitat could improve habitat quality resulting in increased carrying capacity and winter usage by juvenile spring Chinook salmon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: J. A. Cambray     

Cthulhu Macrofasciculumque n. g., n. sp. and Cthylla Microfasciculumque n. g., n. sp., a Newly Identified Lineage of Parabasalian Termite Symbionts

The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species Cthulhu macrofasciculumque and Cthylla microfasciculumque from Prorhinotermes simplex and Reticulitermes virginicus, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. Cthulhu is relatively large and has a distinctive bundle of over 20 flagella whereas Cthylla is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, Cthulhu likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as Tricercomitus.     

A New Algorithm to Diagnose Atrial Ectopic Origin from Multi Lead ECG Systems - Insights from 3D Virtual Human Atria and Torso

Rapid atrial arrhythmias such as atrial fibrillation (AF) predispose to ventricular arrhythmias, sudden cardiac death and stroke. Identifying the origin of atrial ectopic activity from the electrocardiogram (ECG) can help to diagnose the early onset of AF in a cost-effective manner. The complex and rapid atrial electrical activity during AF makes it difficult to obtain detailed information on atrial activation using the standard 12-lead ECG alone. Compared to conventional 12-lead ECG, more detailed ECG lead configurations may provide further information about spatio-temporal dynamics of the body surface potential (BSP) during atrial excitation. We apply a recently developed 3D human atrial model to simulate electrical activity during normal sinus rhythm and ectopic pacing. The atrial model is placed into a newly developed torso model which considers the presence of the lungs, liver and spinal cord. A boundary element method is used to compute the BSP resulting from atrial excitation. Elements of the torso mesh corresponding to the locations of the placement of the electrodes in the standard 12-lead and a more detailed 64-lead ECG configuration were selected. The ectopic focal activity was simulated at various origins across all the different regions of the atria. Simulated BSP maps during normal atrial excitation (i.e. sinoatrial node excitation) were compared to those observed experimentally (obtained from the 64-lead ECG system), showing a strong agreement between the evolution in time of the simulated and experimental data in the P-wave morphology of the ECG and dipole evolution. An algorithm to obtain the location of the stimulus from a 64-lead ECG system was developed. The algorithm presented had a success rate of 93%, meaning that it correctly identified the origin of atrial focus in 75/80 simulations, and involved a general approach relevant to any multi-lead ECG system. This represents a significant improvement over previously developed algorithms.     

Identification of Antibiotic Resistance Cassettes in Class 1 Integrons in Aeromonas spp. Strains Isolated From Fresh Fish (Cyprinus carpio L.)

Forty-six Aeromonas spp. strains were isolated from fresh fish and investigated for their antimicrobial susceptibility, detection of Class 1 integrons by PCR, and arrangement of gene cassettes. Selected isolates were further characterized by enterobacterial repetitive intergenic consensus-PCR. Twenty isolates were found to carry Class 1 integrons. Amplification of the variable regions of the integrons revealed diverse bands ranging in size from 150 to 1,958 pb. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as adenylyl transferases (aadA2 and aadA5), dihydrofolate reductases (dfrA17 and dfrA1), chloramphenicol acetyl transferase (catB3), β-lactamase (oxa2), lincosamide nucleotidil transferase (linF), aminoglycoside-modifying enzyme (apha15), and oxacillinase (bla _OXA-10). Two open reading frames with an unknown function were identified as orfC and orfD. The aadA2 cassette was the most common integron found in this study. Interestingly, five integrons were detected in the plasmids that might be involved in the transfer of resistance genes to other bacteria. This is a first report of cassette encoding for lincosamides (linF) resistance in Aeromonas spp. Implications on the incidence of integrons in isolates of Aeromonas spp. from fresh fish for human consumption, and its possible consequences to human health are discussed.     

Influence of hydrological conditions on the <Emphasis Type="Italic">Escherichia coli</Emphasis> population structure in the water of a creek on a rural watershed

Background  

Escherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. The aim of this study was to investigate the effect of hydrological conditions on the structure of the E. coli population in the water of a creek on a small rural watershed in France composed of pasture and with human occupation.     

Microbial metabolomics: past,present and future methodologies

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSⁿ, GC-MSⁿ, CE-MSⁿ), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).     

The role of farm structure on bird assemblages around a Kenyan tropical rainforest

Although it is clear that the farmlands neighbouring fragmented forests are utilized by some forest birds, it is not clear how birds in general respond to farmland habitat mosaic. An effort was made to determine how bird density and foraging assemblages were influenced by farm structural characteristics and distance from forest edge. Thirty farms up to a distance of 12 km around Kakamega forest in western Kenya were studied. Farm structure entailed size, hedge volume, habitat heterogeneity, woody plant density, plant diversity and crop cover. Birds were surveyed using line transects and DISTANCE analyses and classified into six feeding guilds and three habitat associations. Size of farms increased away from the forest, as woody plant density, plant diversity, indigenous trees and subsistence crop cover declined. The most important farm structure variable was hedge volume, which enhanced bird species richness, richness of shrub‐land bird species and insectivorous bird density (R = 0.58, P < 0.01). Bird density increased with tree density while indigenous trees were suitable for insectivores and nectarivores. There were very few forest bird encounters. Agricultural practices incorporating maintenance of hedges and sound selection of agroforestry trees can enhance conservation of birds on farmland, though, not significantly for forest species.