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.     

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     

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.     

Spatial and temporal factors associated with nest survival of Gray Flycatchers in managed ponderosa pine forests

Gray Flycatchers (Empidonax wrightii) breed in a variety of habitats in the arid and semi‐arid regions of the western United States, but little is known about their breeding biology, especially in the northern portion of their range where they nest in ponderosa pine (Pinus ponderosa) forests. From May to July 2014 and 2015, we conducted surveys for singing male Gray Flycatchers along the eastern slope of the Cascade Range in Washington, U.S.A, monitored flycatcher nests, and quantified nest‐site vegetation. We used a logistic‐exposure model fit within a Bayesian framework to model the daily survival probability of flycatcher nests. During the 2 yr of our study, we monitored 141 nests, with 93% in ponderosa pines. Mean clutch size was 3.6 eggs and the mean number of young fledged per nest was 3.2. Predation accounted for 90% of failed nests. We found a positive association between daily nest survival and both nest height and distance of nest substrates from the nearest tree. Flycatchers that locate their nests higher above the ground and further from adjacent trees may be choosing the safest alternative because higher nests may be less exposed to terrestrial predators and nests in trees that are farther from other trees may be less exposed to arboreal predators such as jays (Corvidae) that may forage in patches with connected canopies. Nests in trees farther from other trees may also allow earlier detection of approaching predators and thus aid in nest defense.     

Microbial Communities Associated with Healthy and White Syndrome-Affected Echinopora lamellosa in Aquaria and Experimental Treatment with the Antibiotic Ampicillin

Prokaryotic and ciliate communities of healthy and aquarium White Syndrome (WS)-affected coral fragments were screened using denaturing gradient gel electrophoresis (DGGE). A significant difference (R = 0.907, p < 0.001) in 16S rRNA prokaryotic diversity was found between healthy (H), sloughed tissue (ST), WS-affected (WSU) and antibiotic treated (WST) samples. Although 3 Vibrio spp were found in WS-affected samples, two of these species were eliminated following ampicillin treatment, yet lesions continued to advance, suggesting they play a minor or secondary role in the pathogenesis. The third Vibrio sp increased slightly in relative abundance in diseased samples and was abundant in non-diseased samples. Interestingly, a Tenacibaculum sp showed the greatest increase in relative abundance between healthy and WS-affected samples, demonstrating consistently high abundance across all WS-affected and treated samples, suggesting Tenacibaculum sp could be a more likely candidate for pathogenesis in this instance. In contrast to previous studies bacterial abundance did not vary significantly (ANOVA, F2, 6 = 1.000, p = 0.422) between H, ST, WSU or WST. Antimicrobial activity (assessed on Vibrio harveyi cultures) was limited in both H and WSU samples (8.1% ±8.2 and 8.0% ±2.5, respectively) and did not differ significantly (Kruskal-Wallis, χ2 (2) = 3.842, p = 0.146). A Philaster sp, a Cohnilembus sp and a Pseudokeronopsis sp. were present in all WS-affected samples, but not in healthy samples. The exact role of ciliates in WS is yet to be determined, but it is proposed that they are at least responsible for the neat lesion boundary observed in the disease.     

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.