José P. Da Silva

Mitotically Stable Association of Polycomb Group Proteins Eed and Enx1 with the Inactive X Chromosome in Trophoblast Stem Cells

X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, which then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Here we show that Eed and a second Polycomb group protein, Enx1, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells.

A photoinduced pH jump applied to drug release from cucurbit[7]uril

A proof-of-principle for the application of a photoinduced pH jump for delivery of the Hoechst 33258 drug by disassembly of its host-guest complex with cucurbit[7]uril is described.

Supramolecular photocatalysis: insights into cucurbit[8]uril catalyzed photodimerization of 6-methylcoumarin

Guest induced shape change of the cucurbit[8]uril cavity is likely rate limiting in the supramolecular photocatalytic cycle for CB8 mediated photodimerization of 6-methylcoumarin.

Aggregates of Cucurbituril Complexes in the Gas Phase

The self-organization of cucurbit[n]uril (n = 7 and 8, CBs) complexes was probed by electrospray mass spectrometry. The self-association of CB complexes is a general phenomenon but shows some dependence on the absence, presence, and type of included guest molecules.

Self aggregation of supramolecules of nitroxides@cucurbit[8]uril revealed by EPR spectra.

Supramolecular complexation behavior of cucurbiturils with paramagnetic nitroxide spin probes was examined by (1)H NMR, X-ray diffraction studies of crystals, computation, and EPR. Both cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) form a 1:1 complex with 4-(N,N,N-trimethylammonium)-2,2,6,6-tetramethylpiperidinyl-N-oxy bromide (CAT1). The structure of the complex in the solid state was inferred by X-ray diffraction studies and in the gas phase by computation (B3LYP/6-31G(d)). Whereas ESI-MS data provided evidence for the existence of the complex in solution, indirect evidence was obtained through (1)H NMR studies with a structural diamagnetic analogue, 4-(N,N,N-trimethylammonium)-2,2,6,6-tetramethyl-N-methylpiperidine iodide (DCAT1). The EPR spectrum of the CAT1@CB7 complex consisting of three lines suggested that probe CAT1 is associated with host CB7 such that the nitroxide part is exposed to water. The spectral pattern was independent of the concentration of the complex and the presence of salt such as NaCl. The most interesting observation was made with CB8 as the host. In this case, in addition to the expected three-line spectrum, an additional spectrum consisting of seven lines was recorded. The contribution of the seven-line spectrum to the total spectrum was dependent on the concentration of the complex and added salt (NaCl) to the aqueous solution. The coupling constant for the seven-line spectrum for (14)N-substituted CAT1 is 5 G, and that for the four-line spectrum for (15)N-substituted CAT1 is 7.15 G. The only manner by which we could reproduce the observed spectra by simulation for both (14)N- and (15)N-substituted CAT1@CB8 was by assuming a spin exchange among three nitroxide radicals. To account for this observation, we hypothesize that three CAT1 molecules included within CB8 interact in such a way that there is an association of three supramolecules of CAT1@CB8 (i.e., [CAT1@CB8](3)) in a triangular geometry that leads to spin exchange between the three radical centers. We have established, with the help of 13 additional examples, that this is a general phenomenon. We are in the process of understanding this unusual phenomenon.

Identity of a Tilapia Pheromone Released by Dominant Males that Primes Females for Reproduction

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11].

Cooperative assembly of discrete stacked aggregates driven by supramolecular host-guest complexation

p-Sulfonatocalix[4]arene (SC4) interacts with the aromatic dye crystal violet (CV) to form complexes with stoichiometries ranging from SC4:CV = 1:1 up to 1:5 both in solution and in the gas phase. While the 1:1 complex is of the inclusion type, as frequently observed for other guests, in the higher-order complexes the CV molecules interact with SC4 in a peripheral manner. The formation of such complexes is driven by ionic interactions established between the dye and the calixarene and by CV-CV stacking interactions. The application of an advanced fitting procedure made possible a quantitative analysis of the UV-vis data and allowed the determination of the stepwise binding constants. This unprecedented approach provides evidence that the formation of the highest-order complexes occurs through a cooperative mechanism. Moreover, the development of a quantitative analytical model enables the possibility of using this type of system for water-soluble sensing assays, as is also exemplified in the present work.

Synthetic versus natural receptors: supramolecular control of chemical sensing in fish.

The encapsulation of odorants by the synthetic receptor cucurbit[7]uril (CB[7]) reduces the response of olfactory receptors in Mozambique tilapia (Oreochromis mossambicus) in vivo. For example, the olfactory receptor response to the odorant adamantan-1-amine, as measured by electro-olfactography, was suppressed by 92% in the presence of CB[7]. A reduction in olfactory response of 88% was observed for pentane-1,5-diamine (cadaverine), an odorant associated with carrion avoidance in some fish. The results reveal how the association constants and the concentrations of natural and synthetic receptors play a determinant role and show that synthetic receptors can be used to remove bioactive molecules from fish olfaction.

Novel laser-induced luminescence resulting from benzophenone/O-propylated p-tert-butylcalix[4]arene complexes. A diffuse reflectance study

Laser-induced room temperature luminescence of air-equilibrated benzophenone/O-propylated p-tert-butylcalix[4]arene solid powdered samples revealed the existence of a novel emission, in contrast with benzophenone/p-tert-butylcalix[4]arene complexes, where only benzophenone emits. This novel emission was identified as phosphorescence of 1-phenyl-1,2-propanedione, which is formed as the result of an hydrogen atom abstraction reaction of the triplet excited benzophenone from the propoxy substituents of the calixarene. Room temperature phosphorescence was obtained in air-equilibrated samples in all propylated hosts. The decay times of the benzophenone emission vary greatly with the degree of propylation, the shortest lifetimes being obtained in the tri- and tetrapropylated calixarenes. Triplet-triplet absorption of benzophenone was detected in all cases, and is the predominant absorption in the p-tert-butylcalix[4]arene case. where an endo-calix complex is formed. Benzophenone ketyl radical formation occurs with the O-propylated p-tert-butylcalix[4]arenes hosts, suggesting a different type of host/guest molecular arrangement. Diffuse reflectance laser flash photolysis and gas chromatography-mass spectrometry techniques provided complementary information, the former about transient species and the latter regarding the final products formed after light absorption. Product analysis and identification clearly show that the two main degradation photoproducts following laser excitation in the propylated substrates are 1-phenyl-1,2-propanedione and 2-hydroxybenzophenone, although several other minor photodegradation products were identified. A detailed mechanistic analysis is proposed. While the solution photochemistry of benzophenone is dominated by the hydrogen abstraction reaction from suitable hydrogen donors, in these solid powdered samples, the alpha-cleavage reaction also plays an important role. This finding occurs even with one single laser pulse which lasts only a few nanoseconds, and is apparently related to the fact that scattered radiation exists, due to multiple internal reflections possibly trapping light within non-absorbing microcrystals in the sample, and is detected until at least 20 micros after the laser pulse. This could explain how photoproducts thus formed could also be excited with only one laser pulse.

Deep-Cavity Cavitand Octa Acid as a Hydrogen Donor: Photofunctionalization with Nitrenes Generated from Azidoadamantanes

1-azidoadamantane and 2-azidoadamantane form a 1:1 complex with hosts octa acid (OA) and cucurbit[7]uril (CB7) in water. Isothermal titration calorimetric measurements suggest these complexes to be very stable in aqueous solution. The complexes have been characterized by (1)H NMR in solution and by ESI-MS in gas phase. In both phases, the complexes are stable. Irradiation of these complexes (λ > 280 nm) results in nitrenes via the loss of nitrogen from the guest azidoadamantanes. The behavior of nitrenes within OA differs from that in solution. Nitrenes included within octa acid attack one of the four tertiary benzylic hydrogens present at the lower interior part of OA. While in solution intramolecular insertion is preferred, within OA intermolecular C-H insertion seems to be the choice. When azidoadamantanes included in CB7 were irradiated (λ > 280 nm) the same products as in solution resulted but the host held them tightly. Displacement of the product required the use of a higher binding guest. In this case, no intermolecular C-H insertion occurred. Difference in reactivity between OA and CB7 is the result of the location of hydrogens; in OA they are in the interior of the cavity where the nitrene is generated, and in CB7 they are at the exterior. Reactivity of nitrenes within OA is different from that of carbenes that do not react with the host.