Rapid Quantification of the Toxic Alga Prymnesium parvum in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Phylogeny of the ectomycorrhizal mushroom genus Alnicola (Basidiomycota, Cortinariaceae) based on rDNA sequences with special emphasis on host specificity and morphological characters

Alnicola (=Naucoria, pro parte) is a mushroom genus of strictly temperate, obligately ectomycorrhizal species, traditionally included in the family Cortinariaceae. Most Alnicola spp. are primarily host specific on Alnus, although a few are mycobionts of Salix or other hosts. The different species of Alnicola exhibit unique morphological (cystidia, pileipellis) and cytological (dikaryotic or monokaryotic hyphae) characters. This makes the genus Alnicola of particular interest for studying the evolution of host specificity and morphological characters in ectomycorrhizal basidiomycetes. We used a combination of classical morphological and phylogenetic methods (rDNA ITS and LSU sequences) to address the following questions: (i) Is Alnicola monophyletic? And (ii) Are characters like host specificity or microscopical structures synapomorphic for certain clades? The study included nearly all currently known European Alnicola sp. Our results demonstrated that, on one hand, the genus Alnicola is polyphyletic, with sistergroup relationships to Hebeloma, Anamika or the clades /Hymenogaster I and /Hymenogaster II. On the other hand, Alnicola splits into three well-supported clades corresponding to the sections Alnicola, Submelinoides, and Salicicolae. The strict host-specificity to Alnus is a derived character and has occurred at least twice. The following morphological characters are synapomorphic for defined clades: the spindle-shaped hymenial cystidia for sect. Alnicola, the hymeniform pileipellis for sect. Submelinoides, and monocaryotic/clampless hyphae for sect. Salicicolae and its sistergroup /Hymenogaster II. As a taxonomical consequence, polyphyly of Alnicola implies that the sects. Submelinoides and Salicicolae need to be segregated from Alnicola.     

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.     

Étude clinique de phase I avec bénéfice individuel direct de radioimmunothérapie du myélome multiple utilisant l’anticorps anti-CD138 marqué à l’iode 131 (131I-B-B4)

PurposeThe objective of this study was to evaluate the toxicity, the absorbed dose to critical organs and tumour of B-B4 monoclonal antibody labeled with Iodine 131 in patients with multiple myeloma (MM) enrolled in a phase I study.Patients and methodFour patients with MM were enrolled and received for dosimetric study an injection of 20 mg/m² of B-B4 coupled with 370 MBq of Iodine 131. During the treatment phase, after viewing the target, three patients received a fixed dose of 20 mg/m² of ¹³¹I-B-B4 with an initial activity of 555 MBq/m², corresponding to level 1.ResultsImmunoscintigraphy showed an early and intensive uptake of the axial skeleton confirming the targeting of the disease by the antibody. Grade 3/4 haematological toxicity was observed in two patients with a trend to tally with the estimated average dose received by the bone marrow, calculated in the dosimetric study (blood method and imaging method). No other toxicity was observed. No complete or partial response was observed.ConclusionThe dose of 555 MBq/m² of ¹³¹I-B-B4 has shown encouraging results in terms of dosimetry and toxicity of RIT in MM. Other developments are possible with the use of humanized monoclonal antibody and the labeling with an alpha particle emitter.     

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.     

Inferring phylogenies from RAD sequence data

Reduced-representation genome sequencing represents a new source of data for systematics, and its potential utility in interspecific phylogeny reconstruction has not yet been explored. One approach that seems especially promising is the use of inexpensive short-read technologies (e.g., Illumina, SOLiD) to sequence restriction-site associated DNA (RAD)--the regions of the genome that flank the recognition sites of restriction enzymes. In this study, we simulated the collection of RAD sequences from sequenced genomes of different taxa (Drosophila, mammals, and yeasts) and developed a proof-of-concept workflow to test whether informative data could be extracted and used to accurately reconstruct "known" phylogenies of species within each group. The workflow consists of three basic steps: first, sequences are clustered by similarity to estimate orthology; second, clusters are filtered by taxonomic coverage; and third, they are aligned and concatenated for "total evidence" phylogenetic analysis. We evaluated the performance of clustering and filtering parameters by comparing the resulting topologies with well-supported reference trees and we were able to identify conditions under which the reference tree was inferred with high support. For Drosophila, whole genome alignments allowed us to directly evaluate which parameters most consistently recovered orthologous sequences. For the parameter ranges explored, we recovered the best results at the low ends of sequence similarity and taxonomic representation of loci; these generated the largest supermatrices with the highest proportion of missing data. Applications of the method to mammals and yeasts were less successful, which we suggest may be due partly to their much deeper evolutionary divergence times compared to Drosophila (crown ages of approximately 100 and 300 versus 60 Mya, respectively). RAD sequences thus appear to hold promise for reconstructing phylogenetic relationships in younger clades in which sufficient numbers of orthologous restriction sites are retained across species.     

Protein misfolding and aggregation in cataract disease and prospects for prevention

The transparency of the eye lens depends on maintaining the native tertiary structures and solubility of the lens crystallin proteins over a lifetime. Cataract, the leading cause of blindness worldwide, is caused by protein aggregation within the protected lens environment. With age, covalent protein damage accumulates through pathways thought to include UV radiation, oxidation, deamidation, and truncations. Experiments suggest that the resulting protein destabilization leads to partially unfolded, aggregation-prone intermediates and the formation of insoluble, light-scattering protein aggregates. These aggregates either include or overwhelm the protein chaperone content of the lens. Here, we review the causes of cataract and nonsurgical methods being investigated to inhibit or delay cataract development, including natural product-based therapies, modulators of oxidation, and protein aggregation inhibitors.