Application of High-Density DNA Resequencing Microarray for Detection and Characterization of Botulinum Neurotoxin-Producing Clostridia

Background

Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs) that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G) and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains.

Methodology/Principal Findings

In the present study, we designed and evaluated the performances of a resequencing microarray (RMA), the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components.This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays.

Conclusions/Significance

The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.     

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore–microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A–overexpressing cancers.     

Phylogenetic analysis ofIridaceae with parsimony and distance methods using the plastid generps4

A molecular phylogeny of the familyIridaceae based on the plastid generps4 was obtained using both parsimony and distance methods. Thirty-four species were examined together with eight outgroup species. Results show that theIridaceae are monophyletic, and thatIsophysis is likely to be the earliest emerging genus. SubfamilyIxioideae plus the generaAristea andNivenia form a strongly supported clade. Within subfam.Iridoideae, the tribeIrideae includes the genusBobartia (of disputed position), and the tribeMariceae includesCypella. The division ofIridoideae into tribes is consistent with their geographical distribution.     

Formation of N-Pyroglutamyl Peptides from N-Glu and N-Gln Precursors in Aplysia Neurons

Abstract : Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry is used to examine the formation of N -pyroglutamate (pGlu) in single, identified neurons from Aplysia . Six pGlu peptides are identified in the R3-14 and the R15 neurons that result from in vivo processing of peptides containing either Glu or Gln at their respective N-termini. Moreover, we show that Glu-derived pGlu is not a sample collection or measurement artifact. The pGlu peptides are detected in isolated cell bodies, regenerated neurites in culture, interganglionic connective nerves, cell homogenates, and collected releasates. We also demonstrate that R3-14 cells readily convert a synthetic N -Glu peptide to its pGlu analogue, indicating the presence of novel enzymatic activity.     

Versatility of the green microalga cell vacuole function as revealed by analytical transmission electron microscopy

Vacuole is a multifunctional compartment central to a large number of functions (storage, catabolism, maintenance of the cell homeostasis) in oxygenic phototrophs including microalgae. Still, microalgal cell vacuole is much less studied than that of higher plants although knowledge of the vacuolar structure and function is essential for understanding physiology of nutrition and stress tolerance of microalgae. Here, we combined the advanced analytical and conventional transmission electron microscopy methods to obtain semi-quantitative, spatially resolved at the subcellular level information on elemental composition of the cell vacuoles in several free-living and symbiotic chlorophytes. We obtained a detailed record of the changes in cell and vacuolar ultrastructure in response to environmental stimuli under diverse conditions. We suggested that the vacuolar inclusions could be divided into responsible for storage of phosphorus (mainly in form of polyphosphate) and those accommodating non-protein nitrogen (presumably polyamine) reserves, respectively.The ultrastructural findings, together with the data on elemental composition of different cell compartments, allowed us to speculate on the role of the vacuolar membrane in the biosynthesis and sequestration of polyphosphate. We also describe the ultrastructural evidence of possible involvement of the tonoplast in the membrane lipid turnover and exchange of energy and metabolites between chloroplasts and mitochondria. These processes might play a significant role in acclimation in different stresses including nitrogen starvation and extremely high level of CO₂ and might also be of importance for microalgal biotechnology. Advantages and limitations of application of analytical electron microscopy to biosamples such as microalgal cells are discussed.     

Cytoplasmic Domain of Sendai Virus HN Protein Contains a Specific Sequence Required for Its Incorporation into Virions

In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.     

c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.     

Design and synthesis of dinucleotide 5'-triphosphates with expanded functionality

We propose the new approach to the synthesis of 5'-triphosphate derivatives of natural and modified dinucleotides with expanded functionality. Our strategy includes the combination of the solution phase synthesis of necessary dimers using the wide range of nucleic acids chemistry methods and the subsequent introduction of the triphosphate residue. A number of the new potential substrates for the template dependent synthesis of nucleic acids with expanded functionality are obtained, namely, 5'-triphosphates of dinucleotides containing the functionally active groups in heterocyclic bases, in carbohydrate-phosphate backbone, and the groups mimicking the residues of natural amino acids. The abilities of the proposed synthetic route are also demonstrated by the synthesis of 5'-triphosphates of dinucleotides with modified carbohydrate-phosphate backbone.     

The Hunneberg Stage (Ordovician) in the area east of St. Petersburg, north-western Russia

The bio- and lithostratigraphy of the poorly known early Ordovician (Arenig) Hunneberg Stage is described from the area east of St. Petersburg (Putilovo Quarry and Lava river), where the Lakity Beds (Leetse Fm.) consist of glauconitic sands and silty clays. The described fauna includes graptolites, conodonts, brachiopods, acritarchs, foraminifers, and several problematic groups. The Lakity Beds are unique within the lower Ordovician sequence of the East Baltic in the abundance of graptolites, co-occurring with conodonts. The conodonts from the lower part of the Lakity Beds belong to the uppermost subzone (Oelandodus elongatus — Acodus deltatus deltatus) of theParoistodus proteus Zone, coinciding with the lower part of theTetragraptus phyllograptoides Zone. This suggests that there is a hiatus between the Lakity Beds and the underlying Nazya Formation. The lower Lakity Beds also contain the brachiopodsLeptembolon lingulaeformis andEosiphonotreta cf.acrotretomorpha, which suggest that they may be correlated with the Joa Member of the Leetse Formation in North Estonia. In the upper part of the Lakity Beds, conodonts of thePrioniodus elegans Zone co-occur with graptolites indicating the upper subzone of theTetragraptus phyllograptoides Zone in the Scandinavian sections. There are still problems of defining and correlating the base of the Billingen Regional Stage, which are outside the scope of this paper.     

RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing

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