343 research outputs found
Re-appearance of antiferromagnetic ordering with Zn and Ni substitution in La_{2-x}Sr_xCuO_4
The effects of nonmagnetic Zn and magnetic Ni substitution for Cu site on
magnetism are studied by measurements of uniform magnetic susceptibility for
lightly doped La_{2-x}Sr_xCu_{1-z}M_zO_4 (M=Zn or Ni) polycrystalline samples.
For the parent x=0, Zn doping suppresses the N\'{e}el temperature T_N whereas
Ni doping hardly changes T_N up to z=0.3. For the lightly doped samples with
T_N~0, the Ni doping recovers T_N. For the superconducting samples, the Ni
doping induces the superconductivity-to-antiferromagnetic transition (or
crossover). All the heavily Ni doped samples indicate a spin glass behavior at
\~15 K.Comment: 2 pages including 3 figures, to be published in Physica C (LT23,
Hiroshima 2002
Effect of interleukins response to ECM-induced acquisition of drug resistance in MCF-7 cells
Aim: To examine the effect of various components of extracellular matrix (ECM) on acquisition of drug resistance to taxol and camptothecin by breast carcinoma cell line MCF-7. Methods: Cancer cells were cultured on bovine serum albumin (BSA), vitronectin (VN), fibronectin (FN), collagen type I (COL-I), or Matrigel-coated plates with or without taxol (paclitaxel) or camptothecin treatment. The effect of anticancer drugs on cell growth was accessed by XTT assay, and the alterations of cellular morphology were examined by phase contrast microscopy. Immunofluorescence study was performed using monoclonal anti-b-tubulin antibody. Results: All cell lines showed a significant decrease in cell survival when treated with anticancer drugs without components of ECM, whereas survival rates of Caco-2, MCF-7 and NCI-H292 were significantly increased when cells were cultured on COL-I- and Matrigel-coated dishes after treatment with paclitaxel or camptothecin. MCF-7 cells showed and maintained a colony formation when cultured on the COL-I- and Matrigel-coated dish. Moreover, cytotoxicity (IC50) was decreased by taxol (paclitaxel) or camptothecin treatment during colony formation in MCF-7 cells, suggesting that morphological changes could increase survival of cells treated with anticancer drugs. Thick circumferential bundles of microtubules around the periphery of the cells and chromatin condensation was not observed for MCF-7 cells on COL-I- and Matrigel-coated dishes treated with paclitaxel. To confirm this, spheroid cells were prepared, and we found that cytotoxicity was decreased for these cells, and significantly increased when cells were co-cultured on Matrigel- or COL-I-coated upper wells. The effect of anticancer drugs on cell survival was efficiently inhibited by interleukin-6 (IL-6) and interleukin-8 (IL-8). Conclusions: Present results suggested that not only integrin-ECM interactions but also other factors such as IL-6 and IL-8 secreted by cancer cells, cultured on COL-I and Matrigel dishes, are involved in the acquisition of drug resistance by MCF-7.Π¦Π΅Π»Ρ: ΠΈΠ·ΡΡΠΈΡΡ Π²Π»ΠΈΡΠ½ΠΈΠ΅ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠΎΠ² Π²Π½Π΅ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠ³ΠΎ ΠΌΠ°ΡΡΠΈΠΊΡΠ° (ECM) Π½Π° ΠΏΡΠΈΠΎΠ±ΡΠ΅ΡΠ΅Π½ΠΈΠ΅ Ρ
ΠΈΠΌΠΈΠΎΡΠ΅Π·ΠΈΡΡΠ΅Π½ΡΠ½ΠΎΡΡΠΈ
ΠΊ ΡΠ°ΠΊΡΠΎΠ»Ρ ΠΈ ΠΊΠ°ΠΌΠΏΡΠΎΡΠ΅ΡΠΈΠ½Ρ ΠΊΠ»Π΅ΡΠΊΠ°ΠΌΠΈ Π»ΠΈΠ½ΠΈΠΈ ΠΊΠ°ΡΡΠΈΠ½ΠΎΠΌΡ ΠΌΠΎΠ»ΠΎΡΠ½ΠΎΠΉ ΠΆΠ΅Π»Π΅Π·Ρ MCF-7. ΠΠ΅ΡΠΎΠ΄Ρ: ΠΊΠ»Π΅ΡΠΊΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π»ΠΈ Π½Π° ΠΏΠ»Π°ΡΠ°Ρ
,
ΠΏΠΎΠΊΡΡΡΡΡ
Π±ΡΡΡΠΈΠΌ ΡΡΠ²ΠΎΡΠΎΡΠΎΡΠ½ΡΠΌ Π°Π»ΡΠ±ΡΠΌΠΈΠ½ΠΎΠΌ (BSA), Π²ΠΈΡΡΠΎΠ½Π΅ΠΊΡΠΈΠ½ΠΎΠΌ (VN), ΡΠΈΠ±ΡΠΎΠ½Π΅ΠΊΡΠΈΠ½ΠΎΠΌ (FN), ΠΊΠΎΠ»Π»Π°Π³Π΅Π½ΠΎΠΌ I ΡΠΈΠΏΠ° (COL-I)
ΠΈΠ»ΠΈ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ, Π±Π΅Π· ΠΈ Ρ Π΄ΠΎΠ±Π°Π²Π»Π΅Π½ΠΈΠ΅ΠΌ ΡΠ°ΠΊΡΠΎΠ»Π° (ΠΏΠ°ΠΊΠ»ΠΈΡΠ°ΠΊΡΠ΅Π») ΠΈΠ»ΠΈ ΠΊΠ°ΠΌΠΏΡΠΎΡΠ΅ΡΠΈΠ½Π°. ΠΠ»ΠΈΡΠ½ΠΈΠ΅ ΠΏΡΠΎΡΠΈΠ²ΠΎΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΡΡ
ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ² Π½Π°
ΡΠΎΡΡ ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈΠ·ΡΡΠ°Π»ΠΈ Ρ ΠΏΠΎΠΌΠΎΡΡΡ XTT-ΡΠ΅ΡΡΠ°, ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΠΊΠ»Π΅ΡΠΎΡΠ½ΠΎΠΉ ΠΌΠΎΡΡΠΎΠ»ΠΎΠ³ΠΈΠΈ ΠΎΡΠΌΠ΅ΡΠ°Π»ΠΈ Π² ΡΠ°Π·ΠΎΠ²ΠΎΠΌ ΠΊΠΎΠ½ΡΡΠ°ΡΡΠ½ΠΎΠΌ ΠΌΠΈΠΊΡΠΎΡΠΊΠΎΠΏΠ΅.
ΠΠΌΠΌΡΠ½ΠΎΡΠ»ΡΠΎΡΠ΅ΡΡΠ΅Π½ΡΠ½ΡΠΌ ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ ΠΎΠΏΡΠ΅Π΄Π΅Π»ΡΠ»ΠΈ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡ Ξ²-ΡΡΠ±ΡΠ»ΠΈΠ½Π°. Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ: Π΄Π»Ρ Π²ΡΠ΅Ρ
ΠΊΠ»Π΅ΡΠΎΡΠ½ΡΡ
Π»ΠΈΠ½ΠΈΠΉ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ
ΡΡΡΠ΅ΡΡΠ²Π΅Π½Π½ΠΎΠ΅ ΡΠ½ΠΈΠΆΠ΅Π½ΠΈΠ΅ Π²ΡΠΆΠΈΠ²Π°Π΅ΠΌΠΎΡΡΠΈ ΠΏΠΎΡΠ»Π΅ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΡ Ρ ΠΏΡΠΎΡΠΈΠ²ΠΎΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΡΠΌΠΈ ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠ°ΠΌΠΈ Π±Π΅Π· ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠΎΠ² ECM, Π² ΡΠΎ
Π²ΡΠ΅ΠΌΡ ΠΊΠ°ΠΊ ΡΡΠΎΠ²Π΅Π½Ρ Π²ΡΠΆΠΈΠ²Π°Π΅ΠΌΠΎΡΡΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ Caco-2, MCF-7 ΠΈ NCI-H292 Π·Π½Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎ Π²ΠΎΠ·ΡΠΎΡ ΠΏΡΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Ρ ΡΠ°ΠΊΡΠΎΠ»ΠΎΠΌ ΠΈΠ»ΠΈ
ΠΊΠ°ΠΌΠΏΡΠΎΡΠ΅ΡΠΈΠ½ΠΎΠΌ Π² ΡΠ°ΡΠΊΠ°Ρ
, ΠΏΠΎΠΊΡΡΡΡΡ
COL-I ΠΈ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ. ΠΠ»Ρ ΠΊΠ»Π΅ΡΠΎΠΊ MCF-7 ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΈ ΡΠΎΡ
ΡΠ°Π½Π΅Π½ΠΈΠ΅ ΠΊΠΎΠ»ΠΎΠ½ΠΈΠΉ ΠΏΡΠΈ
ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Π² ΡΠ°ΡΠΊΠ°Ρ
Ρ COL-I ΠΈ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ. ΠΠΎΠ»Π΅Π΅ ΡΠΎΠ³ΠΎ, ΡΠΈΡΠΎΡΠΎΠΊΡΠΈΡΠ½ΠΎΡΡΡ (IC50) ΡΠ°ΠΊΡΠΎΠ»Π° ΠΈ Π²ΠΎ Π²ΡΠ΅ΠΌΡ ΠΊΠΎΠ»ΠΎΠ½ΠΈΠ΅ΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈΡ
ΠΊΠ»Π΅ΡΠΎΠΊ MCF-7 Π±ΡΠ»Π° ΡΠ½ΠΈΠΆΠ΅Π½Π°, ΡΡΠΎ ΠΏΠΎΠ·Π²ΠΎΠ»ΡΠ΅Ρ ΠΏΡΠ΅Π΄ΠΏΠΎΠ»ΠΎΠΆΠΈΡΡ, ΡΡΠΎ ΠΌΠΎΡΡΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΠΌΠΎΠ³ΡΡ Π²Π»ΠΈΡΡΡ Π½Π° Π²ΡΠΆΠΈΠ²Π°Π΅ΠΌΠΎΡΡΡ
ΠΊΠ»Π΅ΡΠΎΠΊ ΠΏΡΠΈ ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Ρ Ρ
ΠΈΠΌΠΈΠΎΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠΌ. ΠΠ»Ρ ΠΊΠ»Π΅ΡΠΎΠΊ MCF-7, Π²ΡΡΠ°ΡΠΈΠ²Π°Π΅ΠΌΡΡ
Π½Π° ΡΠ°ΡΠΊΠ°Ρ
Ρ COL-I ΠΈ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ, Π½Π΅
ΠΎΡΠΌΠ΅ΡΠ°Π»ΠΈ ΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈΡ ΠΏΠ»ΠΎΡΠ½ΡΡ
ΠΏΠ΅ΡΠΈΡΠ΅ΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠ·Π»ΠΎΠ² ΠΌΠΈΠΊΡΠΎΡΡΡΠ±ΠΎΡΠ΅ΠΊ ΠΈ ΠΊΠΎΠ½Π΄Π΅Π½ΡΠ°ΡΠΈΠΈ Ρ
ΡΠΎΠΌΠ°ΡΠΈΠ½Π°. ΠΠ»Ρ ΠΏΠΎΠ΄ΡΠ²Π΅ΡΠΆΠ΄Π΅Π½ΠΈΡ Π΄Π°Π½Π½ΠΎΠ³ΠΎ
Π½Π°Π±Π»ΡΠ΄Π΅Π½ΠΈΡ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½Ρ ΠΎΠΏΡΡΡ Ρ ΠΊΠ»Π΅ΡΠΊΠ°ΠΌΠΈ, ΡΠ°ΡΡΡΡΠΈΠΌΠΈ Π² Π²ΠΈΠ΄Π΅ ΡΡΠ΅ΡΠΎΠΈΠ΄ΠΎΠ². ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΡΠΈΡΠΎΡΠΎΠΊΡΠΈΡΠ½ΠΎΡΡΡ Ρ
ΠΈΠΌΠΈΠΎΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ² ΠΏΠΎ
ΠΎΡΠ½ΠΎΡΠ΅Π½ΠΈΡ ΠΊ ΡΡΠΈΠΌ ΠΊΠ»Π΅ΡΠΊΠ°ΠΌ ΡΠ½ΠΈΠΆΠ°Π»Π°ΡΡ ΠΈ Π·Π½Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎ ΠΏΠΎΠ²ΡΡΠ°Π»Π°ΡΡ ΠΏΡΠΈ ΠΊΠΎ-ΠΊΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Ρ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ ΠΈΠ»ΠΈ COL-I Π² Π²Π΅ΡΡ
Π½ΠΈΡ
ΠΊΠ°ΠΌΠ΅ΡΠ°Ρ
. Π‘Π½ΠΈΠΆΠ΅Π½ΠΈΠ΅ Π²ΡΠΆΠΈΠ²Π°Π΅ΠΌΠΎΡΡΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ ΠΏΠΎΠ΄ Π΄Π΅ΠΉΡΡΠ²ΠΈΠ΅ΠΌ Ρ
ΠΈΠΌΠΈΠΎΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ² ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΠΎ ΠΈΠ½Π³ΠΈΠ±ΠΈΡΠΎΠ²Π°Π»ΠΎΡΡ ΠΈΠ½ΡΠ΅ΡΠ»Π΅ΠΉΠΊΠΈΠ½ΠΎΠΌ-6
(IL-6) ΠΈ ΠΈΠ½ΡΠ΅ΡΠ»Π΅ΠΉΠΊΠΈΠ½ΠΎΠΌ-8 (IL-8). ΠΡΠ²ΠΎΠ΄Ρ: Π½Π°ΡΡΠΎΡΡΠΈΠ΅ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΠΏΠΎΠΊΠ°Π·Π°Π»ΠΈ, ΡΡΠΎ Π½Π΅ ΡΠΎΠ»ΡΠΊΠΎ ΠΈΠ½ΡΠ΅Π³ΡΠΈΠ½-ECM-Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ,
Π½ΠΎ ΡΠ°ΠΊΠΆΠ΅ ΠΈ Π΄ΡΡΠ³ΠΈΠ΅ ΡΠ°ΠΊΡΠΎΡΡ, ΡΠ°ΠΊΠΈΠ΅ ΠΊΠ°ΠΊ IL-6 ΠΈ IL-8, ΡΠ΅ΠΊΡΠ΅ΡΠΈΡΡΠ΅ΠΌΡΠ΅ ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΡΠΌΠΈ ΠΊΠ»Π΅ΡΠΊΠ°ΠΌΠΈ Π½Π° ΡΠ°ΡΠΊΠ°Ρ
Ρ COL-I ΠΈ ΠΌΠ°ΡΡΠΈΠ³Π΅Π»Π΅ΠΌ,
ΡΡΠ°ΡΡΠ²ΡΡΡ Π² ΠΏΡΠΈΠΎΠ±ΡΠ΅ΡΠ΅Π½ΠΈΠΈ Ρ
ΠΈΠΌΠΈΠΎΡΠ΅Π·ΠΈΡΡΠ΅Π½ΡΠ½ΠΎΡΡΠΈ ΠΎΠΏΡΡ
ΠΎΠ»Π΅Π²ΡΠΌΠΈ ΠΊΠ»Π΅ΡΠΊΠ°ΠΌΠΈ MCF-7
Interleukin-6 gene (IL-6): a possible role in brain morphology in the healthy adult brain
Background: Cytokines such as interleukin 6 (IL-6) have been implicated in dual functions in neuropsychiatric disorders. Little is known about the genetic predisposition to neurodegenerative and neuroproliferative properties of cytokine genes. In this study the potential dual role of several IL-6 polymorphisms in brain morphology is investigated. Methodology: In a large sample of healthy individuals (Nβ=β303), associations between genetic variants of IL-6 (rs1800795; rs1800796, rs2069833, rs2069840) and brain volume (gray matter volume) were analyzed using voxel-based morphometry (VBM). Selection of single nucleotide polymorphisms (SNPs) followed a tagging SNP approach (e.g., Stampa algorigthm), yielding a capture 97.08% of the variation in the IL-6 gene using four tagging SNPs. Principal findings/results: In a whole-brain analysis, the polymorphism rs1800795 (β174βC/G) showed a strong main effect of genotype (43 CC vs. 150 CG vs. 100 GG; xβ=β24, yβ=ββ10, zβ=ββ15; F(2,286)β=β8.54, puncorrectedβ=β0.0002; pAlphaSim-correctedβ=β0.002; cluster size kβ=β577) within the right hippocampus head. Homozygous carriers of the G-allele had significantly larger hippocampus gray matter volumes compared to heterozygous subjects. None of the other investigated SNPs showed a significant association with grey matter volume in whole-brain analyses. Conclusions/significance: These findings suggest a possible neuroprotective role of the G-allele of the SNP rs1800795 on hippocampal volumes. Studies on the role of this SNP in psychiatric populations and especially in those with an affected hippocampus (e.g., by maltreatment, stress) are warranted.Bernhard T Baune, Carsten Konrad, Dominik Grotegerd, Thomas Suslow, Eva Birosova, Patricia Ohrmann, Jochen Bauer, Volker Arolt, Walter Heindel, Katharina Domschke, Sonja SchΓΆning, Astrid V Rauch, Christina Uhlmann, Harald Kugel and Udo Dannlowsk
Impacts of introduced species on the biota of an oceanic archipelago: the relative importance of competitive and trophic interactions
Expression of osterix Is Regulated by FGF and Wnt/Ξ²-Catenin Signalling during Osteoblast Differentiation
Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways.
Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical
Wingless-type MMTV integration site (Wnt/Ξ²-Catenin) signalling pathways on zebrafish
osteogenesis. We have used transgenic and chemical interference approaches to manipulate
these pathways and have found that both pathways are required for osteoblast differentiation
in vivo. Our analysis of bone markers suggests that these pathways act at the same
stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix
(osx). We use two independent approaches that suggest that osx is a direct target of these
pathways. Firstly, we manipulate signalling and show that osx gene expression responds
with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways.
Secondly, we have performed ChIP with transcription factors for both pathways and our
data suggest that a genomic region in the first intron of osx mediates transcriptional activation.
Based upon these data, we propose that FGF and Wnt/Ξ²-Catenin pathways act in part
by directing transcription of osx to promote osteoblast differentiation at sites of bone
formation
The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa
<p>Abstract</p> <p>Background</p> <p>The cosmopolitan freshwater snail <it>Physa acuta </it>has recently found widespread use as a model organism for the study of mating systems and reproductive allocation. Mitochondrial DNA phylogenies suggest that <it>Physa carolinae</it>, recently described from the American southeast, is a sister species of <it>P. acuta</it>. The divergence of the <it>acuta/carolinae </it>ancestor from the more widespread <it>P. pomilia </it>appears to be somewhat older, and the split between a hypothetical <it>acuta/carolinae/pomilia </it>ancestor and <it>P. gyrina </it>appears older still.</p> <p>Results</p> <p>Here we report the results of no-choice mating experiments yielding no evidence of hybridization between <it>gyrina </it>and any of four other populations (<it>pomilia, carolinae</it>, Philadelphia <it>acuta</it>, or Charleston <it>acuta</it>), nor between <it>pomilia </it>and <it>carolinae</it>. Crosses between <it>pomilia </it>and both <it>acuta </it>populations yielded sterile F1 progeny with reduced viability, while crosses between <it>carolinae </it>and both <it>acuta </it>populations yielded sterile F1 hybrids of normal viability. A set of mate-choice tests also revealed significant sexual isolation between <it>gyrina </it>and all four of our other <it>Physa </it>populations, between <it>pomilia </it>and <it>carolinae</it>, and between <it>pomilia </it>and Charleston <it>acuta</it>, but not between <it>pomilia </it>and the <it>acuta </it>population from Philadelphia, nor between <it>carolinae </it>and either <it>acuta </it>population. These observations are consistent with the origin of hybrid sterility prior to hybrid inviability, and a hypothesis that speciation between <it>pomilia </it>and <it>acuta </it>may have been reinforced by selection for prezygotic reproductive isolation in sympatry.</p> <p>Conclusions</p> <p>We propose a two-factor model for the evolution of postzygotic reproductive incompatibility in this set of five <it>Physa </it>populations consistent with the Dobzhansky-Muller model of speciation, and a second two-factor model for the evolution of sexual incompatibility. Under these models, species trees may be said to correspond with gene trees in American populations of the freshwater snail, <it>Physa</it>.</p
Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting
BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA
Identification of a sex-linked SNP marker in the salmon louse (Lepeophtheirus salmonis) using RAD sequencing
The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a parasitic copepod that can, if untreated, cause considerable damage to Atlantic salmon (Salmo salar Linnaeus, 1758) and incurs significant costs to the Atlantic salmon mariculture industry. Salmon lice are gonochoristic and normally show sex ratios close to 1:1. While this observation suggests that sex determination in salmon lice is genetic, with only minor environmental influences, the mechanism of sex determination in the salmon louse is unknown. This paper describes the identification of a sex-linked Single Nucleotide Polymorphism (SNP) marker, providing the first evidence for a genetic mechanism of sex determination in the salmon louse. Restriction site-associated DNA sequencing (RAD-seq) was used to isolate SNP markers in a laboratory-maintained salmon louse strain. A total of 85 million raw Illumina 100 base paired-end reads produced 281,838 unique RAD-tags across 24 unrelated individuals. RAD marker Lsa101901 showed complete association with phenotypic sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay for genotyping, this SNP association pattern was further confirmed for three unrelated salmon louse strains, displaying complete association with phenotypic sex in a total of 96 genotyped individuals. The marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. This study's observations of a novel sex-linked SNP marker are consistent with sex determination in the salmon louse being genetic and following a female heterozygous system. Marker Lsa101901 provides a tool to determine the genetic sex of salmon lice, and could be useful in the development of control strategies
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