Detection of nitrate/nitrite bioavailability in wastewater using a luxCDABE-based Klebsiella oxytoca bioluminescent bioreporter

In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate (R2 = 0.98), 1 to 6 ppm for nitrite (R2 = 0.99), and 1 to 7 ppm for ammonium (R2 = 0.99). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.     

Tissue Distribution of Anidulafungin in Neonatal Rats

Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4–8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography‐tandem mass spectrometry. The mean plasma C_max and AUC_0‐24 values were consistent with single‐dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone‐to‐plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3‐ to 1.8‐fold. Brain concentrations were low after single dose, with brain‐to‐plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single‐dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.     

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

Background

Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.

Results

Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.

Conclusion

This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.     

Towards a reliable objective function for multiple sequence alignments.

Multiple sequence alignment is a fundamental tool in a number of different domains in modern molecular biology, including functional and evolutionary studies of a protein family. Multiple alignments also play an essential role in the new integrated systems for genome annotation and analysis. Thus, the development of new multiple alignment scores and statistics is essential, in the spirit of the work dedicated to the evaluation of pairwise sequence alignments for database searching techniques. We present here norMD, a new objective scoring function for multiple sequence alignments. NorMD combines the advantages of the column-scoring techniques with the sensitivity of methods incorporating residue similarity scores. In addition, norMD incorporates ab initio sequence information, such as the number, length and similarity of the sequences to be aligned. The sensitivity and reliability of the norMD objective function is demonstrated using structural alignments in the SCOP and BAliBASE databases. The norMD scores are then applied to the multiple alignments of the complete sequences (MACS) detected by BlastP with E-value<10, for a set of 734 hypothetical proteins encoded by the Vibrio cholerae genome. Unrelated or badly aligned sequences were automatically removed from the MACS, leaving a high-quality multiple alignment which could be reliably exploited in a subsequent functional and/or structural annotation process. After removal of unreliable sequences, 176 (24 %) of the alignments contained at least one sequence with a functional annotation. 103 of these new matches were supported by significant hits to the Interpro domain and motif database.     

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission

During transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.     

Synthesis of stereospecifically deuterated phenylalamines and determination of their configuration.

1. Starting from trans-cinnamic acid a chiral (-)3-phenyl-[2,3-2H]propionic acid has been synthesized using Clostridium kiuyveri cells as catalyst. 2. The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)-phenyl[2,3-2H]-alanine. 3. By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L-amino acid oxidase the configuration of the phenylalamine has been shown to be (2R, 3S) and (2S, 3S), respectively. The labelled phenylalanine is thus sterically and isotopically homogenous at position 3 but heterogenous at position 2.     

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane.

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.     

Substrate recognition by a sucrose transporting protein

Protoplasts derived from developing soybean cotyledons were used to study substrate recognition by a sucrose transporting protein in plant membranes. When used as alternate substrate inhibitors of [14C] sucrose influx, five different fructosyl-substituted sucrose derivatives, phenyl-alpha-D-glucopyranoside, and phenyl-alpha-D-thioglucopyranoside proved to bind effectively to the sucrose carrier-active site. These results are interpreted to indicate that a large portion of substrate recognition by this carrier may arise from the interaction of a relatively hydrophobic portion of the sucrose molecule and a hydrophobic region of the carrier protein binding site. Binding of phenyl-alpha-D-thioglucopyranosides in which various substitutions were made for the glucosyl hydroxyls shows that the glucosyl hydroxyls at positions 3, 4, and 6 are involved in substrate recognition by the carrier protein.     

Stereospecificity of phenylpyruvate tautomerase. A convenient method for the preparation of chirally labelled phenylpyruvates.

1. By 1H nuclear magnetic resonance spectroscopy it has been shown that phenylpyruvate tautomerase from beef kidney catalyses the stereospecific exchange of one of the enantiotopic 3H-atoms in the side-chain of the substrate with solvent protons, the rate of spontaneous exchange being slow under physiological conditions. 2. Using monodeuterated phenylpyruvate of known absolute configuration it has been shown that the tautomerase removes specifically the 3-pro-R hydrogen atom of the substrate. 3. The circular dichroism spectra of the two enantiomeric 3-phenyl-[3-2H]pyruvates have been determined. 4. Some implications of these findings for the investigation of the metabolism of aromatic amino acids are discussed.     

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

Background

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.

Methodology/Principal Findings

Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH₂) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH₂ supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.

Conclusions/Significance

The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.