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.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

PHENOTYPIC PLASTICITY INDUCED IN TRANSPLANT EXPERIMENTS IN A MUTUALISTIC ASSOCIATION BETWEEN THE RED ALGA JANIA ADHAERENS (RHODOPHYTA,CORALLINALES) AND THE SPONGE HALICLONA CAERULEA (PORIFERA: HAPLOSCLERIDA): MORPHOLOGICAL RESPONSES OF THE ALGA1

The association between the red macroalga Jania adhaerens J. V. Lamour. and the sponge Haliclona caerulea is the most successful life‐form between 2 and 4 m depth in Mazatlán Bay (Mexican Pacific). J. adhaerens colonizes the rocky intertidal area and penetrates into deeper areas only when it lives in association with H. caerulea. The aposymbiotic form of the sponge has not been reported in the bay. To understand the ecological success of this association, we examined the capacity of J. adhaerens to acclimate in Mazatlán Bay using transplant experiments. The transplanted aposymbiotic J. adhaerens did not survive the first 2 weeks; however, J. adhaerens when living in association with H. caerulea, acclimated easily to depth, showing no sign of mortality during the 103 d of the experiment. We conclude that the ability of J. adhaerens to colonize in deeper areas in this hydrodynamic environment may in part rely on the protection provided by the sponge to the algal canopy. Both species contribute to the shape of the associated form. Nevertheless, the morphological variation in the association appears to be dominated by the variation in J. adhaerens canopy to regulate pigment self‐shading under light‐limited conditions and/or tissue resistance under high hydrodynamics. Consequently, our results are consistent with light as the abiotic controlling factor, which regulates the lower depth distribution of the association in Mazatlán Bay, through limiting the growth rate of J. adhaerens. Hydrodynamics may determine the upper limit of the association by imposing high mass losses.     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Advances in the systematics and evolution of western Mediterranean representative species of the Pentanema conyzae clade through genetic fingerprinting

Forty-five populations of Pentanema corresponding to seven species included in the Pentanema conyzae clade have been studied using AFLP fingerprinting. The results show that allopolyploidization could have been involved in the diversification of this group, specifically in species P. langeanum and P. maletii. Molecular data confirm the presence of P. britannicum in the Iberian Peninsula and key steps are provided to identify the species that are morphologically the most challenging.     

THE EFFECT OF GROWTH ILLUMINATION ON THE PIGMENTATION OF A MARINE DINOFLAGELLATE12

Amphidinium klebsii cultures grown under different light intensities exhibited similar chlorophyll a content per cell. Among the accessory pigments, chlorophyll c concentration decreased slightly in cells exposed to increasing light intensities up to 0.129 ly/min. The concentration of the 2 major xanthophylls present in A. klebsii cells–peridinin and diadinoxanthin–however, varied according to the light background of the cells. Some biochemical pathways in the formation of peridinin in dinoflagellates are discussed.     

Deep-sea squat lobster biogeography (Munidopsidae: Leiogalathea) unveils Tethyan vicariance and evolutionary patterns shared by shallow-water relatives

The ecology, abundance and diversity of galatheoid squat lobsters make them an ideal group to study deep-sea diversification processes. Here, we reconstructed the evolutionary and biogeographic history of Leiogalathea, a genus of circum-tropical deep-sea squat lobsters, in order to compare patterns and processes that have affected shallow-water and deep-sea squat lobster species. We first built a multilocus phylogeny and a calibrated species tree with a relaxed clock using StarBEAST2 to reconstruct evolutionary relationships and divergence times among Leiogalathea species. We used BioGeoBEARS and a DEC model, implemented in RevBayes, to reconstruct ancestral distribution ranges and the biogeographic history of the genus. Our results showed that Leiogalathea is monophyletic and comprises four main lineages; morphological homogeneity is common within and between clades, except in one; the reconstructed ancestral range of the genus is in the Atlantic and Indian oceans (Tethys). They also revealed the divergence of the Atlantic species around 25 million years ago (Ma), intense cladogenesis 15–25 Ma and low levels of speciation over the last 5 million years (Myr). The four Leiogalathea lineages showed similar patterns of speciation: allopatric speciation followed by range expansion and subsequent stasis. Leiogalathea started diversifying during the Oligocene, likely in the Tethyan. The Atlantic lineage then split from its Indo-Pacific sister group due to vicariance driven by closure of the Tethys Seaway. The Atlantic lineage is less speciose compared with the Indo-Pacific lineages, with the Tropical Southwestern Pacific being the current centre of diversity. Leiogalathea diversification coincided with cladogenetic peaks in shallow-water genera, indicating that historical biogeographic events similarly shaped the diversification and distribution of both deep-sea and shallow-water squat lobsters.     

Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions

Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.