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.     

Vegetation fluctuation in mediterranean dune ponds in relation to rainfall variation and water extraction

Abstract. The structure of dune ponds hygrophytic vegetation has a spatial organisation in belts around the pond basin, closely related to water level and flooding regime. Doñana National Park has an important representation of temporal dune ponds, which are subjected to rainfall fluctuations and may be suffering the impact of water extraction from the neighbouring tourist resort. Permanent transects in a vegetation complex of five dune ponds have been monitored during a eight year period (1990-1997). This period was characterised by a number of dry years (annual rainfall around 300 mm), located between two wet cycles (800-900 mm). Transects were analysed in 1990 (wet period), 1994 (dry) and 1997 (wet) by hierarchical agglomera-tive clustering. During the dry period hygrophytic species showed regression, with a high mortality of some species like Ulex minor, while the xerophytic species advanced to lower areas. Seedlings of some xerophytic species colonised the dry surface of the pond basin. The lowering of the water table varied in the different ponds, ranging from 312 to 190 cm depending on topography and the distance to the pumping area. The new period of flooding during 1995-96 and 1996-97 cycles provided the opportunity for hygrophytic spe cies to re-establish themselves in their original places. This study suggest that changes in vegetation are caused by the interaction between weather conditions and human disturbance (water extractions). In our example man-made disturbance is more marked during the dry periods while wet periods tend to obscure the effects of water extractions. From a management perspective, long-term monitoring of water table and vegetation structure is revealed as a key procedure to the management of land-water ecotones on pressured areas and threatened habitats.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Na+/K+-ATPase Inhibition Partially Mimics the Ethanol-Induced Increase of the Golgi Cell-Dependent Component of the Tonic GABAergic Current in Rat Cerebellar Granule Cells

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I_tonic) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I_tonic that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I_tonic in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I_tonic and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na⁺/K⁺-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23–30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I_tonic that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I_tonic. Inhibition of the Na⁺/K⁺-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na⁺/K⁺-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system.     

Habitat preference in the critically endangered yellow‐tailed woolly monkey (Lagothrix flavicauda) at La Esperanza,Peru

Habitat loss is one of the main threats to wildlife. Therefore, knowledge of habitat use and preference is essential for the design of conservation strategies and identification of priority sites for the protection of endangered species. The yellow‐tailed woolly monkey (Lagothrix flavicauda Humboldt, 1812), categorized as Critically Endangered on the IUCN Red List, is endemic to montane forests in northern Peru where its habitat is greatly threatened. We assessed how habitat use and preference in L. flavicauda are linked to forest structure and composition. The study took place near La Esperanza, in the Amazonas region, Peru. Our objective was to identify characteristics of habitat most utilized by L. flavicauda to provide information that will be useful for the selection of priority sites for conservation measures. Using presence records collected from May 2013 to February 2014 for one group of L. flavicauda, we classified the study site into three different use zones: low‐use, medium‐use, and high‐use. We assessed forest structure and composition for all use zones using 0.1 ha Gentry vegetation transects. Results show high levels of variation in plant species composition across the three use zones. Plants used as food resources had considerably greater density, dominance, and ecological importance in high‐use zones. High‐use zones presented similar structure to medium‐ and low‐use zones; thus it remains difficult to assess the influence of forest structure on habitat preference. We recommend focusing conservation efforts on areas with a similar floristic composition to the high‐use zones recorded in this study and suggest utilizing key alimentation species for reforestation efforts.     

Behavioural modification of a social spider by a parasitoid wasp

1. Manipulation of host behaviour by parasitoids has long captured the imagination of ecologists. Parasitoid wasps in the Polysphincta group of genera develop as external parasitoids of spiders. 2. In the present study, the previously undescribed interaction between a Zatypota sp. wasp (Ichneumonidae) and a social spider Anelosimus eximius (Theridiidae) is described. The larva of this Zatypota wasp is found to induce its host to disperse from their communal web and build an entirely enclosed web consisting of densely spun silk. 3. The wasp is observed to target primarily immature A. eximius individuals, with 37.5–44% of nests in a given area being parasitised. Of those nests, approximately 1.3–2.0% of individuals are hosts to the parasitoid larvae. Larger spider colonies had a significantly higher probability of harbouring parasitoids. 4. This interaction results in unusual behaviours for A. eximius induced by the parasitoid: (i) leaving the protection of the social nest and (ii) building a unique, altered web that it would not otherwise build. It is suggested that the wasp may be tapping into ancestral dispersal behaviours in its host and that a social species provides this wasp an evolutionary advantage by allowing a stable host source.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Intrauterine programming of ageing

Ageing is an unavoidable corollary to being alive; the most intuitive interpretation of ageing being that it is the consequence of progressive body degeneration. In agreement with this, current models propose that ageing occurs through a stepwise accumulation of DNA damage, which ultimately limits the regenerative capacity of tissues. On the other hand, there is increasing evidence that fetal distress can influence the development of disease in adult life, a phenomenon known as ‘intrauterine programming’. The extent to which an intrauterine exposure to DNA damage can compromise lifespan remains unclear. My group has recently generated a murine model of a human syndrome linked to defective DNA repair and observed that these animals age prematurely, but the accumulation of DNA damage is restricted mostly to the embryonic period. Here, I discuss the implications of this finding and propose that ageing can be influenced by fetal distress.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.     

Development of the visual system of anchovy larvae,Engraulis anchoita: A microanatomical description

During the early ontogeny of fish larvae, the accurate development of the visual system plays a key role, because it is involved in locating food, orientation, selection of favorable habitat, and evasion of predators. The structure of the eye of the fish is typical of vertebrates, with some modifications related to the aquatic environment. In the present work, we describe the development of the larval eye of Engraulis anchoita for the first time. Larvae were collected at the Permanent Station of Environmental Studies (EPEA) in coastal waters of the Southwestern Atlantic Ocean during research cruises in 2015 and 2016. We describe the histology of the retina layers, determine the beginning of the functionality of the eye, and discuss a possible synchronization with the development of the digestive tract. This study provides information about the biology of E. anchoita, the most abundant fish species in the southwestern Atlantic Ocean. Also, recent studies have shown responses of the retina and other tissues to the increase in environmental acidity. Therefore, results of this study are also discussed with respect to the possible effect of acidification on the larvae of this species. The continuity of the time series developed at the EPEA will allow monitoring the effect of long-term environmental and biological variables on the early ontogeny of anchovy in the context of climate change. The high commercial fishing potential of E. anchoita due to its high abundance, as well as its essential role in the trophic web of other commercially valuable fishing resources of Argentina, reinforce the need to continue deepening knowledge about this species. Research highlights:

• Eyes of Engraulis anchoita larvae are functional from early larval stages.
• At hatching, the retina is formed by only few layers from which the other layers differentiates during ontogeny.
• Focal distance increases with larval growth.