On the applicability of discrete dipole approximation for plasmonic particles

It has been recognized that the commonly used discrete dipole approximation (DDA) for calculating the optical properties of plasmonic materials may exhibit slow convergence for a certain region of the complex refractive index. In this work we investigate the quantitative accuracy of DDA for particles of different shapes, with silver as the plasmonic material. As expected, the accuracy and convergence of the method as a function of the number of dipoles is relatively good for solid spheres and rounded cubes whose size is of the same order as the wavelength of the localized surface plasmon resonance in silver. However, we find that for solid particles much smaller than the resonance wavelength, and for silver-silica core-shell particles in particular, DDA does not converge to the correct limit even for 10(6) dipoles. We also find that the slow convergence tends to be accompanied by strong, discretization dependent oscillations in the particle's internal electric field. We demonstrate that the main factor behind the slow convergence of the DDA is due to inaccuracies in the plasmonic resonances of the dipoles at the surface of the particles. (C) 2015 Elsevier Ltd. All rights reserved.Peer reviewe

Multiplexing with three-primer PCR for rapid and economical microsatellite validation

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci

Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA

Phylogenetic Relationships of the Marine Haplosclerida (Phylum Porifera) Employing Ribosomal (28S rRNA) and Mitochondrial (cox1, nad1) Gene Sequence Data

The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here

Development and application of molecular tools for the study of farmed and wild Atlantic cod (Gadus morhua) stocks

Atlantic cod (Gadus morhua) is a commercially and ecologically important fish inhabiting the shelf areas of the North Atlantic. Several genetic resources were developed for the study of cod in this thesis. Fifty-five new microsatellite loci in six multiplex panels were developed for characterising the wild Celtic Sea stock. The existing methodology for validation of novel microsatellite markers was improved by combining three-primer-PCR with multiplex reactions. In addition, the potential of microsatellite genotyping-by-sequencing (GBS) was investigated using a combinatorial barcoding method suitable for implementation in large-scale population genetics studies. The results confirmed a high correspondence between GBS and capillary/gel electrophoresis genotypes, but also revealed cases of homoplasy. Genetic structuring of local wild cod in waters surrounding Ireland was investigated and a baseline of genetic variation was recorded for wild cod from Celtic Sea, Irish Sea and West of Ireland using 53 microsatellite markers. The results showed that cod from the above areas likely form a genetically homogenous population. Genetic markers were employed to monitor the composition of an early phase breeding programme. Parentage analysis revealed large differences in survival between families during rearing for all three sampled year classes (2008, 2009 and 2010). These differences in survival resulted in large variation in family size which was a major contributing factor to a very low effective population size in the farmed stock for the sampled year classes. In addition, F1 broodstock fish contributing to mass spawning events were shown to have unequal reproductive contributions to the F2 cohort. These findings highlight the importance of genetic monitoring in early-phase breeding programs, shed light on the population structure of cod in Irish waters and provide new tools for both gadoid research and other research questions that can be addressed by means of microsatellite marker variation.2017-05-3

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding

This study examines the potential of next-generation sequencing based 'genotyping-by-sequencing' (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding.

This study examines the potential of next-generation sequencing based 'genotyping-by-sequencing' (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data..S.V. and R.F. acknowledge funding from the Sea Change Strategy with the support of the Marine Institute and the Marine Research Sub-programme of the National Development Plan 2007–2013, co-financed by the European Regional Development Fund (Grant-Aid Agreement no. PBA/AF/07/004, EIRCOD). J.L.V.-C. acknowledges funding from the Spanish Ministerio de Economía y Competitividad (FPI BES-2010–038494 and EEBB-I-13–06270). G.H. acknowledges funding by the Irish Research Council (IRC) Graduate Research Education Programme (GREP). P.M. and J.C. acknowledge funding from the Beaufort Marine Research Award in Fish Population Genetics funded by the Irish Government under the Sea Change Programme. P.C.C. acknowledges funding from Science Foundation Ireland (SFI 12/IP/1308). E.D.F. acknowledges funding from Irish Research Council (IRC) (Funding Agency Ref no./nGOIPD/2013/320)