In vitro regeneration and optimization of factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation in <Emphasis Type="Italic">Artemisia Pallens</Emphasis>, an important medicinal plant

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog’s medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog’s medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD₆₀₀ of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T₀ transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.     

An Efficient Multiple-Testing Adjustment for eQTL Studies that Accounts for Linkage Disequilibrium between Variants

Methods for multiple-testing correction in local expression quantitative trait locus (cis-eQTL) studies are a trade-off between statistical power and computational efficiency. Bonferroni correction, though computationally trivial, is overly conservative and fails to account for linkage disequilibrium between variants. Permutation-based methods are more powerful, though computationally far more intensive. We present an alternative correction method called eigenMT, which runs over 500 times faster than permutations and has adjusted p values that closely approximate empirical ones. To achieve this speed while also maintaining the accuracy of permutation-based methods, we estimate the effective number of independent variants tested for association with a particular gene, termed M_eff, by using the eigenvalue decomposition of the genotype correlation matrix. We employ a regularized estimator of the correlation matrix to ensure M_eff is robust and yields adjusted p values that closely approximate p values from permutations. Finally, using a common genotype matrix, we show that eigenMT can be applied with even greater efficiency to studies across tissues or conditions. Our method provides a simpler, more efficient approach to multiple-testing correction than existing methods and fits within existing pipelines for eQTL discovery.     

Analyzing Photothermal Heat Generation Efficiency in a Molecular Plasmonic Silver Nanomatryushka Dimer

We report on the numerically and analytically investigated plasmonic and photothermal responses of a nanomatryushka structure composed of silver concentric nanoshells which exhibited strong plasmon resonance localization in the optical frequencies. Illuminating an isolated silver nanomatryushka in an aqueous system, we calculated the photothermal response of the structure and quantified the absorbed optical power and generated photothermal heat. In addition, it is shown that a couple of nanomatryushka structures as a symmetric molecular dimer in weak and strong coupling regimes are able to support strong plasmon resonances in the visible to the near-infrared region. Utilizing strong near-field coupling in the metallic nanostructures and hybridization of plasmons, and also employing silver as a highly absorptive material at the visible spectrum, we increased the energy dissipation per unit volume almost three orders of magnitude in comparison to the other analogous subwavelength structures. Employing numerical methods, we showed that a symmetric metallic nanomatryushka dimer is able to generate enough photothermal heat which could result in a remarkable amount of temperature change (ΔT?=?140 K) at the picosecond time scale. According to hybridization theory, the symmetric dimer is able to support strong bonding and antibonding plasmon resonant modes. Utilizing concentric nanoshells with high geometrical tunability facilitates using all of the surfaces and center of nanoparticles to generate heat with a large temperature change within a short relaxation time. This understanding opens new avenues to utilize simple nanoparticle orientations to generate significant heat power in an extremely short time scale for cancer therapy, photothermal therapy, and biological applications.     

Molecular barcoding and phylogeography of sexual and asexual freshwater planarians of the genus Dugesia in the Western Mediterranean (Platyhelminthes, Tricladida, Dugesiidae)

Planarians of the genus Dugesia have a worldwide distribution with high species diversity in the Mediterranean area. In this area, populations with a triploid karyotype that reproduce by fissiparity are exceptionally frequent, outnumbering the sexual populations. This situation poses interesting questions, such as the age of these asexual lineages, whether they all belong to the same species or whether the triploidization event is recurrent, and what factors (climatic, geographical, historical...) explain the prevalence of these asexual forms. However, asexual populations cannot be assigned to a species due to the lack of copulatory apparatus – the main structure used in species identification. In this study, we have developed a DNA barcoding method, based on COI and ITS-1 sequences, which allows the assignment of the fissiparous forms to sexual species. At the same time, phylogenetic analysis from species of the western Mediterranean have unveiled the presence of species with highly differentiated populations alongside species with a wide distribution and almost no genetic variation. The roles of habitat instability, dispersal capacity and human activities are briefly discussed.     

Riboflavin binding protein contains a type II copper binding site

Riboflavin binding protein, purified from egg white, binds copper(II) under dialysis conditions in an approximately 1:1 molar ratio. Results further indicate a small, but not negligible, amount of copper is present in the protein as purified from egg white. Electron paramagnetic resonance indicates a single type II copper site present in the protein. These results suggest the possibility of a previously unknown function of riboflavin binding protein in the storage or transport of copper.     

Mitochondrial DNA signals of late glacial recolonization of Europe from near eastern refugia

Human populations, along with those of many other species, are thought to have contracted into a number of refuge areas at the height of the last Ice Age. European populations are believed to be, to a large extent, the descendants of the inhabitants of these refugia, and some extant mtDNA lineages can be traced to refugia in Franco-Cantabria (haplogroups H1, H3, V, and U5b1), the Italian Peninsula (U5b3), and the East European Plain (U4 and U5a). Parts of the Near East, such as the Levant, were also continuously inhabited throughout the Last Glacial Maximum, but unlike western and eastern Europe, no archaeological or genetic evidence for Late Glacial expansions into Europe from the Near East has hitherto been discovered. Here we report, on the basis of an enlarged whole-genome mitochondrial database, that a substantial, perhaps predominant, signal from mitochondrial haplogroups J and T, previously thought to have spread primarily from the Near East into Europe with the Neolithic population, may in fact reflect dispersals during the Late Glacial period, ～19-12 thousand years (ka) ago.     

Mitogenomes from Two Uncommon Haplogroups Mark Late Glacial/Postglacial Expansions from the Near East and Neolithic Dispersals within Europe

The current human mitochondrial (mtDNA) phylogeny does not equally represent all human populations but is biased in favour of representatives originally from north and central Europe. This especially affects the phylogeny of some uncommon West Eurasian haplogroups, including I and W, whose southern European and Near Eastern components are very poorly represented, suggesting that extensive hidden phylogenetic substructure remains to be uncovered. This study expanded and re-analysed the available datasets of I and W complete mtDNA genomes, reaching a comprehensive 419 mitogenomes, and searched for precise correlations between the ages and geographical distributions of their numerous newly identified subclades with events of human dispersal which contributed to the genetic formation of modern Europeans. Our results showed that haplogroups I (within N1a1b) and W originated in the Near East during the Last Glacial Maximum or pre-warming period (the period of gradual warming between the end of the LGM, ∼19 ky ago, and the beginning of the first main warming phase, ∼15 ky ago) and, like the much more common haplogroups J and T, may have been involved in Late Glacial expansions starting from the Near East. Thus our data contribute to a better definition of the Late and postglacial re-peopling of Europe, providing further evidence for the scenario that major population expansions started after the Last Glacial Maximum but before Neolithic times, but also evidencing traces of diffusion events in several I and W subclades dating to the European Neolithic and restricted to Europe.