Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species

Low- versus Mid-frequency Raman Spectroscopy for in Situ Analysis of Crystallization in Slurries

Slurry studies are useful for exhaustive polymorph and solid-state stability screening of drug compounds. Raman spectroscopy is convenient for monitoring crystallization in such slurries, as the measurements can be performed in situ even in aqueous environments. While the mid-frequency region (400-4000 cm(-1)) is dominated by intramolecular vibrations and has traditionally been used for such studies, the low-frequency spectral region (Peer reviewe

Understanding Dissolution and Crystallization with Imaging: A Surface Point of View

The tendency for crystallization during storage and administration is the most considerable hurdle for poorly water-soluble drugs formulated in the amorphous form. There is a need to better detect often subtle and complex surface crystallization phenomena and understand their influence on the critical quality attribute of dissolution. In this study, the interplay between surface crystallization of the amorphous form during storage and dissolution testing, and its influence on dissolution behavior, is analyzed for the first time with multimodal nonlinear optical imaging (coherent anti-Stokes Raman scattering (CARS) and sum frequency generation (SFG)). Complementary analyses are provided with scanning electron microscopy, X-ray diffraction and infrared and Raman spectroscopies. Amorphous indomethacin tablets were prepared and subjected to two different storage conditions (30 °C/23% RH and 30 °C/75% RH) for various durations and then dissolution testing using a channel flow-through device. Trace levels of surface crystallinity previously imaged with nonlinear optics after 1 or 2 days of storage did not significantly decrease dissolution and supersaturation compared to the freshly prepared amorphous tablets while more extensive crystallization after longer storage times did. Multimodal nonlinear optical imaging of the tablet surfaces after 15 min of dissolution revealed complex crystallization behavior that was affected by both storage condition and time, with up to four crystalline polymorphs simultaneously observed. In addition to the well-known α- and γ-forms, the less reported metastable ε- and η-forms were also observed, with the ε-form being widely observed in samples that had retained significant surface amorphousness during storage. This form was also prepared in the pure form and further characterized. Overall, this study demonstrates the potential value of nonlinear optical imaging, together with more established solid-state analysis methods, to understand complex surface crystallization behavior and its influence on drug dissolution during the development of amorphous drugs and dosage forms.Peer reviewe

Estrogen-inducible and liver-specific expression of the chicken very low density apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal

Surface stabilization and dissolution rate improvement of amorphous compacts with thin polymer coatings: can we have it all?

The distinction between surface and bulk crystallization of amorphous pharmaceuticals, as well as the importance of surface crystallization for pharmaceutical performance, is becoming increasingly evident. An emerging strategy in stabilizing the amorphous drug form is to utilize thin coatings at the surface. While the physical stability of systems coated with pharmaceutical polymers has recently been studied, the effect on dissolution performance as a function of storage time, as a further necessary step toward the success of these formulations, has not been previously studied. Furthermore, the effect of coating thickness has not been elucidated. This study investigated the effect of these polymer-coating parameters on the interplay between amorphous surface crystallization and drug dissolution for the first time. The study utilized simple tablet-like coated dosage forms, comprising a continuous amorphous drug core and thin polymer coating (hundreds of nanometers to a micrometer thick). Monitoring included analysis of both the solid-state of the model drug (with SEM, XRD, and ATR FTIR spectroscopy) and dissolution performance (and associated morphology and solid-state changes) after different storage times. Stabilization of the amorphous form (dependent on the coating thickness) and maintenance of early-stage intrinsic dissolution rates characteristic for the unaged amorphous drug were achieved. However, dissolution in the latter stages was likely inhibited by the presence of a polymer at the surface. Overall, this study introduced a versatile coated system for studying the dissolution of thin-coated amorphous dosage forms suitable for different drugs and coating agents. It demonstrated the importance of multiple factors that need to be taken into consideration when aiming to achieve both physical stability and improved release during the shelf life of amorphous formulations.Peer reviewe

Cell-Nanoparticle Interactions at (Sub)-Nanometer Resolution Analyzed by Electron Microscopy and Correlative Coherent Anti-Stokes Raman Scattering

A wide variety of nanoparticles are playing an increasingly important role in drug delivery. Label-free imaging techniques are especially desirable to follow the cellular uptake and intracellular fate of nanoparticles. The combined correlative use of different techniques, each with unique advantages, facilitates more detailed investigation about such interactions. The synergistic use of correlative coherent anti-Stokes Raman scattering and electron microscopy (C-CARS-EM) imaging offers label-free, chemically-specific, and (sub)-nanometer spatial resolution for studying nanoparticle uptake into cells as demonstrated in the current study. Coherent anti-Stokes Raman scattering (CARS) microscopy offers chemically-specific (sub)micron spatial resolution imaging without fluorescent labels while transmission electron microscopy (TEM) offers (sub)-nanometer scale spatial resolution and thus visualization of precise nanoparticle localization at the sub-cellular level. This proof-of-concept imaging platform with unlabeled drug nanocrystals and macrophage cells revealed good colocalization between the CARS signal and electron dense nanocrystals in TEM images. The correlative TEM images revealed subcellular localization of nanocrystals inside membrane bound vesicles, showing multivesicular body (MVB)-like morphology typical for late endosomes (LEs), endolysosomes, and phagolysosomes. C-CARS-EM imaging has much potential to study the interactions between a wide range of nanoparticles and cells with high precision and confidence.Peer reviewe

Importance of the Active Site "Canopy" Residues in an O_2-Tolerant [NiFe]-Hydrogenase

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a “canopy” above the bimetallic center, closest to the site at which exogenous agents CO and O_2 interact, substrate H_2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H_2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H_2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H_2/D_2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H_2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H_2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O_2 tolerance by stabilizing the oxidized resting Ni^(III)–OH state (“Ni-B”). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe

ChemDiverse : a chemistry careers activity showcasing diversity

Women, ethnic minority and less affluent groups are widely under-represented in chemistry, a problem that is observed at all levels but begins before college matriculation takes place. The importance of representation and humanization of scientists is crucial. Despite limited progress over recent decades poor visibility of role models from under-represented groups remains problematic, emphasizing the importance of initiatives to positively introduce them in classroom settings. Through profiles of under-represented “success stories” from academia and industry, the ChemDiverse project was developed to encourage under-represented groups to pursue the chemical sciences at higher education level by providing teachers with an easy and structured way of encouraging Scottish high school students into Science, technology, engineering, and mathematics (STEM) higher education. Based on survey feedback from teachers at participating schools, it is a well-formulated project which is easy to implement within the context of the Scottish Curriculum for Excellence

Chiral Polymerization in Open Systems From Chiral-Selective Reaction Rates

We investigate the possibility that prebiotic homochirality can be achieved exclusively through chiral-selective reaction rate parameters without any other explicit mechanism for chiral bias. Specifically, we examine an open network of polymerization reactions, where the reaction rates can have chiral-selective values. The reactions are neither autocatalytic nor do they contain explicit enantiomeric cross-inhibition terms. We are thus investigating how rare a set of chiral-selective reaction rates needs to be in order to generate a reasonable amount of chiral bias. We quantify our results adopting a statistical approach: varying both the mean value and the rms dispersion of the relevant reaction rates, we show that moderate to high levels of chiral excess can be achieved with fairly small chiral bias, below 10%. Considering the various unknowns related to prebiotic chemical networks in early Earth and the dependence of reaction rates to environmental properties such as temperature and pressure variations, we argue that homochirality could have been achieved from moderate amounts of chiral selectivity in the reaction rates.Comment: 15 pages, 6 figures, accepted for publication in Origins of Life and Evolution of Biosphere