Gas chromatographic method for the determination of lumefantrine in antimalarial finished pharmaceutical products

A simple method has been developed and validated for quantitative determination of lumefantrine in antimalarial finished pharmaceutical products using gas chromatography coupled to flame ionization detector. Lumefantrine was silylated with N,O–bis(trimethyl-silyl)trifluoro-acetamide at 70°C for 30 minutes, and chromatographic separation was conducted on a fused silica capillary (HP-5, 30 m length × 0.32 mm i.d., 0.25 μm film thickness) column. Evaluation of the method within analytical quality-by-design principles, including a central composite face-centered design for the sample derivatization process and Plackett–Burman robustness verification of the chromatographic conditions, indicated that the method has acceptable specificity toward excipients and degradants, accuracy [mean recovery = 99.5%, relative standard deviation (RSD) = 1.0%], linearity (=0.9986), precision (intraday = 96.1% of the label claim, RSD = 0.9%; interday = 96.3% label claim, RSD = 0.9%), and high sensitivity with detection limits of 0.01 μg/mL. The developed method was successfully applied to analyze the lumefantrine content of marketed fixed-dose combination antimalarial finished pharmaceutical products

Location and dynamics of an active promoter in Escherichia coli K-12

In the present paper, we report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor–GFP (green fluorescent protein) fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned constitutive promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression

Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor

Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies

Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

<p>Abstract</p> <p>Background</p> <p>The <it>Burkholderia cepacia </it>complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511).</p> <p>Results</p> <p>KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the <it>Peduovirinae </it>subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 <it>E+E' </it>translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The <it>lysBC </it>genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an IS<it>Bmu</it>2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations.</p> <p>Conclusions</p> <p>KS5, KS14 and KL3 are the first BCC-specific phages to be identified as P2-like. As KS14 has previously been shown to be active against <it>Burkholderia cenocepacia in vivo</it>, genomic characterization of these phages is a crucial first step in the development of these and similar phages for clinical use against the BCC.</p

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities

pGIL01, a linear tectiviral plasmid prophage from Bacillus thuringiensis serovar israelensis

The Bacillus cereus sensu lato group includes six closely related bacteria, namely B. cereus sensu stricto, B. thuringiensis, B. anthracis, B. weihenstephanensis, B. mycoides and B. pseudomycoides. B. thuringiensis is an entomopathogenic strain used worldwide as bio-insecticide to control insect pests. More specifically, B. thuringiensis serovar israelensis is active against lepidopteran larvae, such as black flies and mosquitoes, two important vectors of human and animal diseases. The plasmid pattern analysis of B. thuringiensis serovar israelensis strain 4Q2-72 revealed the presence of at least eight plasmids, including four large and three small circular plasmids, as well as one unconventional linear element of 15kb. This linear molecule was reported for the first time in 1984 by Gonzàlez and Carlton but so far, no characterization of this small linear plasmid has been undertaken. The primary objective of the present work was therefore to establish a first description of this linear element, designated pGIL01, in order to get more insights concerning its contribution into the plasmid biology of B. thuringiensis. The first part of this thesis consisted in the genetic description of this linear molecule pGIL01. Its sequence analysis, as well as experimental evidences, revealed that pGIL01 is the prophage form of a temperate phage, named GIL01, that belonged to the Tectiviridae family. Members of this group are characterized by the presence of lipid components in their capsids. Both the plasmid prophage and the viral genomes possess the same linear ds DNA molecule of 14,931 bp protected by proteins at their 5'extremities and with imperfect terminal inverted repeats of 73 bp at both ends. These data establish, for the first time, the existence of a prophage existing as a linear element harbouring terminal proteins. The activity of two putative peptidoglycan hydrolases identified on (p)GIL01 have been experimentally confirmed. While both lytic enzymes are able to cleave Gram-positive peptidoglycan preparations, only one is active against Gram-negative bacteria. The second part of this study focused on the occurrence of (p)GIL01-related linear elements among the B. cereus sensu lato group. The genetic comparison of (p)GIL01 with three other (putative) Gram-positive tectiviruses (Bam35, GIL01 and pBClin15) and with the Gram-negative tectivirus PRD1 revealed that the Tectiviridae family is prone to extensive genetic flexibility.Doctorat en sciences (sciences biologiques) (BIOL 3)--UCL, 200