Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ∼10,000 years before present (YBP), with signals of expansions at ∼3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈15%) of the Franco-Cantabrian maternal gene pool with a putative pre-Neolithic origin to ≈35%, further supporting the notion of a predominant Paleolithic genetic substrate in extant European populations.     

Effect of dilution rate on eicosapentaenoic acid productivity ofPhaeodactylum tricornutum utex 640 in outdoor chemostat culture

Summary Eicosapentaenoic acid (EPA) volumetric productivity from an outdoor chemostat culture ofPhaeodactylum tricornutum UTEX 640 in a 50-l tubular photobioreactor varies with dilution rate, reaching a maximum of 47.8 mg l^–1 d^–1 at D=0.36 d^–1. Continuous culture at high dilution rates' is proposed as the most adequate operating mode to maximize polyunsaturated fatty acid production.     

Biogeography of Iberian freshwater fishes revisited: the roles of historical versus contemporary constraints

Aim The question of how much of the shared geographical distribution of biota is due to environmental vs. historical constraints remains unanswered. The aim of this paper is to disentangle the contribution of historical vs. contemporary factors to the distribution of freshwater fish species. In addition, it illustrates how quantifying the contribution of each type of factor improves the classification of biogeographical provinces. Location Iberian Peninsula, south‐western Europe (c. 581,000 km²). Methods We used the most comprehensive data on native fish distributions for the Iberian Peninsula, compiled from Portuguese and Spanish sources on a 20‐km grid‐cell resolution. Overall, 58 species were analysed after being categorized into three groups according to their ability to disperse through saltwater: (1) species strictly intolerant of saltwater (primary species); (2) species partially tolerant of saltwater, making limited incursions into saltwaters (secondary species); and (3) saltwater‐tolerant species that migrate back and forth from sea to freshwaters or have invaded freshwaters recently (peripheral species). Distance‐based multivariate analyses were used to test the role of historical (basin formation) vs. contemporary environmental (climate) conditions in explaining current patterns of native fish assemblage composition. Cluster analyses were performed to explore species co‐occurrence patterns and redefine biogeographical provinces based on the distributions of fishes. Results River basin boundaries were better at segregating species composition for all species groups than contemporary climate variables. This historical signal was especially evident for primary and secondary freshwater fishes. Eleven biogeographical provinces were delineated. Basins flowing to the Atlantic Ocean north of the Tagus Basin and those flowing to the Mediterranean Sea north of the Mijares Basin were the most dissimilar group. Primary and secondary freshwater species had higher province fidelity than peripheral species. Main conclusions The results support the hypothesis that historical factors exert greater constraints on native freshwater fish assemblages in the Iberian Peninsula than do current environmental factors. After examining patterns of assemblage variation across space, as evidenced by the biogeographical provinces, we discuss the likely dispersal and speciation events that underlie these patterns.     

Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

Role of Asp-9 and Glu-36 in the active site of the pneumococcal CPL1 lysozyme: an evolutionary perspective of lysozyme mechanism.

The role of carboxylic amino acids Asp-9 and Glu-36 in the activity of CPL1 lysozyme was investigated by site-directed mutagenesis. The enzymatic activity of the single mutants D9E, D9N, D9H, D9K, D9A, E36D, E36Q, E36K, and E36A and of the double mutant D9A-E36A was analyzed using a highly sensitive radioactive assay. All mutants but D6K showed detectable activities. Interestingly, the mutants E36D and E36Q retained 67% and 37% activity, respectively. Amino acid replacements at position 9 turned out to be more critical for activity than at position 36. In analogy to the mechanism described for hen egg-white lysozyme, where the proton donor play a central role, we propose that, in the CPL1 lysozyme, Asp-9 might act as the proton donor for activation of the substrate, and Glu-36 could help in the stabilization of the intermediate oxocarbocation. The residual activity of lysozyme mutants lacking one or two of the acidic amino acids may be explained by the participation of a water molecule as proton donor and/or to electrostatic contributions in the active center stabilizing the transition state of the reaction. Our results are in agreement with the hypothesis that enzymes have been optimized during evolution from an ancestral protein able to bind more tightly the transition state of the substrate than the substrate itself, by the acquisition of amino acids serving a function in catalysis.     

Associated Liver Partition and Portal Vein Ligation (ALPPS) vs Selective Portal Vein Ligation (PVL) for Staged Hepatectomy in a Rat Model. Similar Regenerative Response?

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two-stage hepatectomy technique which can be associated with a hypertrophic stimulus on the future liver remnant (FLR) stronger than other techniques–such as portal vein ligation (PVL). However, the reason of such hypertrophy is still unclear, but it is suggested that liver transection combined with portal vein ligation (ALPPS) during the first stage of this technique may play a key role. The aim of this study is to compare the hypertrophic stimulus on the FLR and the clinical changes associated with both ALPPS and PVL in a rat surgical model. For this purpose, three groups of SD rats were used, namely ALPPS (n = 30), PVL (n = 30) and sham-treated (n = 30). The second stage of ALPPS (hepatectomy of the atrophic lobes), was performed at day 8. Blood and FLR samples were collected at 1, 24, 48 hours, 8 days and 12 weeks after the surgeries. ALPPS provoked a greater degree of hypertrophy of the FLR than the PVL at 48 hours and 8 days (p<0.05). The molecular pattern was also different, with the highest expression of IL-1β at 24h, IL-6 at 8 days, and HGF and TNF-α at 48 hours and 8 days (p<0.05). ALPPS also brought about a mild proliferative stimulus at 12 weeks, with a higher expression of HGF and TGF-β (p<0.05) than PVL. Clinically, ALPPS caused a significant liver damage during the first 48 hours, with a recovery of liver function at day 8. In conclusion, ALPPS seems to induce higher functional hypertrophy on the FLR than PVL at day 8. Such regenerative response seems to be leaded by a complex interaction between pro-mitogenic (IL-6, HGF, TNF-α) and antiproliferative (IL1-β and TGF-β) cytokines.     

Combination of Hansen Robotic system with cryocatheter in a challenging parahisian accessory pathway ablation

A perceived distinctive feature of cryoablation is the stability (cryoadherence) of the catheter tip during cold temperatures at the desired location, even during tachycardia. We report the case report of a young patient with a parahisian accessory pathway where stability of the ablation catheter was not achieved despite using the cryocatheter with a steerable sheath. Ultimately, stability at the desired location was achieved robotically by means of Hansen system (Hansen Medical, Mountain View, CA, USA).