Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

Tularemia in a common marmoset (Callithrix jacchus) diagnosed by 16S rRNA sequencing

We report a case of tularemia in a common marmoset (Callithrix jacchus) diagnosed by determination of the isolate's 16S ribosomal RNA (rRNA) gene sequence. Pathological examination of the animal revealed a multifocal acute necrotizing hepatitis, interstitial nephritis, splenitis, and lymphangitis of the mandibular, retropharyngeal, and cervical and mesenteric lymph nodes. Moreover, multiple foci of acute necrosis were found in the epithelium of the jejunum and the interstitium of the lung. Bacteriological investigations revealed a septicemia. The isolated infectious agent was uncommon, not routinely diagnosed in our laboratory and therefore difficult to identify by conventional tools in a reasonable time and effort. thus, we decided to perform a genetic analysis based on the 16S rRNA gene sequence. Thereby, an infection with Francisella tularensis, the causative agent of tularemia, was unambiguously diagnosed. This shows the great advantage 16S rRNA gene sequencing has as a general identification approach for unusual or rare isolates.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Environmental conditions and habitats of GOS sites and bottle data of HOT-ALOHA station

The Global Ocean Sampling (GOS) expedition is currently the largest and geographically most comprehensive metagenomic dataset, including samples from the Atlantic, Pacific, and Indian Oceans. This study makes use of the wide range of environmental conditions and habitats encompassed within the GOS sites in order to investigate the ecological structuring of bacterial and archaeal taxon ranks. Community structures based on taxonomically classified 16S ribosomal RNA (rRNA) gene fragments at phylum, class, order, family, and genus rank levels were examined using multivariate statistical analysis, and the results were inspected in the context of oceanographic environmental variables and structured habitat classifications. At all taxon rank levels, community structures of neritic, oceanic, estuarine biomes, as well as other exotic biomes (salt marsh, lake, mangrove), were readily distinguishable from each other. A strong structuring of the communities with chlorophyll a concentration and a weaker yet significant structuring with temperature and salinity were observed. Furthermore, there were significant correlations between community structures and habitat classification. These results were used for further investigation of one-to-one relationships between taxa and environment and provided indications for ecological preferences shaped by primary production for both cultured and uncultured bacterial and archaeal clades.

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5′ sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids of Marsilea

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while β-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea β-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither β-tubulin- nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita.

Detecção e genotipagem de astrovírus em amostras fecais de aves de produção procedentes de diferentes criações comerciais brasileiras

Os astrovírus são agentes virais associados com enteropatias e infecções extra-intestinais em aves, ocasionando prejuízos no desenvolvimento produtivo e saúde animal. Porém, são escassos os estudos no Brasil que visem a detecção e caracterização deste vírus com maior amplitude. Nesse contexto, detectaram-se e caracterizaram-se cepas de astrovírus a partir de 60 amostras fecais de aves procedentes de diferentes criações comerciais brasileiras (postura, corte e matrizes), mediante o emprego de uma reação PanAstrovírus como triagem inicial, que consistiu numa reação de RT-hemi-nested-PCR, visando a amplificação e posterior sequenciamento de um segmento parcial do gene RdRp (RNA polimerase dependente de RNA) do gene ORF1b, uma região gênica conservada entre todos os astrovírus. Subsequentemente, nas amostras positivas na triagem, realizou-se a amplificação, clonagem e sequenciamento do gene ORF2 codificante do capsídeo viral, analisado mediante análise filogenética. Os dados obtidos demonstraram uma frequência de infecção por astrovírus em 48,3% (29/60) das amostras analisadas. As espécies virais detectadas foram as seguintes: Avastrovírus 2 (Avian Nephritis Virus, ANV) em 72,4% (21/29), Chicken Astrovírus (CAstV) em 6,8% (2/29) e Avastrovírus 1 (Turkey Astrovírus tipo 1, TAstV-1) em 20,8% (6/29) das amostras positivas. O sequenciamento total do gene ORF2 foi possível em oito amostras (8/21) positivas a ANV, em uma amostra (1/6) positiva a TAstV-1 e uma amostra (1/2) positiva para CAstV. A análise deste gene revelou, nas sequências ANV, a presença de três genótipos bem diferenciados, segundo critérios do Astroviridae Study Group, sendo que um deles, agrupou-se dentro do genótipo 5. Além disso, quando analisado com sequências procedentes de outras regiões, as sequências deste estudo formaram seis clusters, dois deles, relacionados com sequências procedentes de Austrália e Reino Unido. O análise do ORF2 em CAstV, revelou também um agrupamento com cepas procedentes do Reino Unido. Já o análise da ORF2 de TAstV-1, revelou divergência genética com cepas isoladas em Estados Unidos, que foram as únicas sequências disponíveis no GenBank. O presente estudo traz a luz vários dados epidemiológicos concernentes aos astrovírus aviário de origem brasileira o que permitirá a realização de outros estudos relacionados a epidemiologia deste vírus, seus possíveis riscos potenciais em saúde pública e a adoção de medidas de controle direcionadas a este agente

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Evolution of resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum

The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosage, time of treatment, and drug elimination half-life, with an in-host dynamics model of P. falciparum malaria in a malaria-naive host. The results indicate that the development of drug resistance can be categorized into three stages. The first is the selection of existing parasites with genetic mutations in the dihydrofolate reductase or dihydropteroate synthetase gene. This selection is driven by the long half-life of the sulfadoxine-pyrimethamine combination. The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection. The timing of treatment relative to initiation of a specific anti-P. falciparum EMP1 immune response is an important factor during this stage, as is the treatment dosage. During the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutations to survive therapeutic doses of the drug combination. Therefore, the model output reaffirms the importance of correct treatment of confirmed malaria cases in slowing the development of parasite resistance to sulfadoxine-pyrimethamine.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Tracking the shift to 'Postgenomics'

Current knowledge about the variety and complexity of the processes that allow regulated gene expression in living organisms calls for a new understanding of genes. A 'postgenomic' understanding of genes as entities constituted during genome expression is outlined and illustrated with specific examples that formed part of a survey research instrument developed by two of the authors for an ongoing empirical study of conceptual change in contemporary biology. Copyright (C) 2006 S. Karger AG, Basel.

Sequence analysis of complementary DNAs encoding C3 in the nurse shark ({\it Ginglymostoma cirratum\/})

Mammalian C3 is a pivotal complement protein, encoded for by a single gene. In some vertebrate species multiple C3 isoforms are products of different C3 genes. The goal of this study was to determine whether multiple genes encode for shark C3. A protocol was developed for the isolation of mRNA from shark blood for the isolation of C3 cDNA clones. RT-PCR amplification of mRNA, using sense (GCGEQNM) and antisense (TWLTAYV) primers encoding conserved regions of human C3, yielded 21 clones. The C3-like clones isolated shared 97% similarity with each other and 40% similarity to human C3. RACE-PCR amplification of shark liver RNA, using gene specific primers, yielded products ranging from 1800bp to 3000bp. Deduced amino acid sequence, corresponding to 408bp of the 1800bp fragment, was obtained which showed 51% similarity to human C3. These results suggest that nurse shark C3 might be encoded for by more than one gene. ^

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.