Neutron scattering study of polyamorphic THF·17(H2O) - toward a generalized picture of amorphous states and structures derived from clathrate hydrates.

From crystalline tetrahydrofuran clathrate hydrate, THF-CH (THF·17H2O, cubic structure II), three distinct polyamorphs can be derived. First, THF-CH undergoes pressure-induced amorphization when pressurized to 1.3 GPa in the temperature range 77-140 K to a form which, in analogy to pure ice, may be called high-density amorphous (HDA). Second, HDA can be converted to a densified form, VHDA, upon heat-cycling at 1.8 GPa to 180 K. Decompression of VHDA to atmospheric pressure below 130 K produces the third form, recovered amorphous (RA). Results from neutron scattering experiments and molecular dynamics simulations provide a generalized picture of the structure of amorphous THF hydrates with respect to crystalline THF-CH and liquid THF·17H2O solution (∼2.5 M). Although fully amorphous, HDA is heterogeneous with two length scales for water-water correlations (less dense local water structure) and guest-water correlations (denser THF hydration structure). The hydration structure of THF is influenced by guest-host hydrogen bonding. THF molecules maintain a quasiregular array, reminiscent of the crystalline state, and their hydration structure (out to 5 Å) constitutes ∼23H2O. The local water structure in HDA is reminiscent of pure HDA-ice featuring 5-coordinated H2O. In VHDA, the hydration structure of HDA is maintained but the local water structure is densified and resembles pure VHDA-ice with 6-coordinated H2O. The hydration structure of THF in RA constitutes ∼18 H2O molecules and the water structure corresponds to a strictly 4-coordinated network, as in the liquid. Both VHDA and RA can be considered as homogeneous.

The impact of temperature and unwanted impurities on slow compression of ice.

For slowly compressed hexagonal ice pressure-induced amorphisation to high-density amorphous ice (HDA) takes place below and at 130 K, but polymorphic transformation to ice IX takes place at 140-170 K. Stable ice II only forms above 170 K. Ice IX impurities trigger ice IX growth even at 120 K. HDA and ice IX are equally long-lived, where both can be regarded as metastable phases.

Small-angle neutron scattering study of micropore collapse in amorphous solid water.

Vapor-deposited amorphous solid water (ASW) is the most abundant solid molecular material in space, where it plays a direct role in both the formation of more complex chemical species and the aggregation of icy materials in the earliest stages of planet formation. Nevertheless, some of its low temperature physics such as the collapse of the micropore network upon heating are still far from being understood. Here we characterize the nature of the micropores and their collapse using neutron scattering of gram-quantities of D2O-ASW of internal surface areas up to 230 ± 10 m(2) g(-1) prepared at 77 K. The model-free interpretation of the small-angle scattering data suggests micropores, which remain stable up to 120-140 K and then experience a sudden collapse. The exact onset temperature to pore collapse depends on the type of flow conditions employed in the preparation of ASW and, thus, the specific surface area of the initial deposit, whereas the onset of crystallization to cubic ice is unaffected by the flow conditions. Analysis of the small-angle neutron scattering signal using the Guinier-Porod model suggests that a sudden transition from three-dimensional cylindrical pores with 15 Å radius of gyration to two-dimensional lamellae is the mechanism underlying the pore collapse. The rather high temperature of about 120-140 K of micropore collapse and the 3D-to-2D type of the transition unraveled in this study have implications for our understanding of the processing and evolution of ices in various astrophysical environments.

Integrative nucleophosmin mutation-associated microRNA and gene expression pattern analysis identifies novel microRNA - target gene interactions in acute myeloid leukemia.

BACKGROUND: MicroRNAs are regulators of gene expression, which act mainly by decreasing mRNA levels of their multiple targets. Deregulated microRNA expression has been shown for acute myeloid leukemia, a disease also characterized by altered gene expression associated with distinct genomic aberrations such as nucleophosmin (NPM1) mutations. To shed further light on the role of deregulated microRNA and gene expression in cytogenetically normal acute myeloid leukemia with NPM1 mutation we performed an integrative analysis of microRNA and mRNA expression data sets. DESIGN AND METHODS: Both microRNA and gene expression profiles were investigated in samples from a cohort of adult cytogenetically normal acute myeloid leukemia patients (n=43; median age 46 years, range 23-60 years) with known NPM1 mutation status (n=23 mutated, n=20 wild-type) and the data were integratively analyzed. Putative microRNA-mRNA interactions were validated by quantitative reverse transcriptase polymerase chain reaction, western blotting and luciferase reporter assays. For selected microRNAs, sensitivity of microRNA-overexpressing cells to cytarabine treatment was tested by FACS viability and cell proliferation assays. RESULTS: Our integrative approach of analyzing both microRNA- and gene expression profiles in parallel resulted in a refined list of putative target genes affected by NPM1 mutation-associated microRNA deregulation. Of 177 putative microRNA - target mRNA interactions we identified and validated 77 novel candidates with known or potential involvement in leukemogenesis, such as IRF2-miR-20a, KIT-miR-20a and MN1-miR-15a. Furthermore, our data showed that deregulated expression of tumor suppressor microRNAs, such as miR-29a and miR-30c, might contribute to sensitivity to cytarabine, which is observed in NPM1 mutated acute myeloid leukemia. CONCLUSIONS: Overall, our observations highlight that integrative data analysis approaches can improve insights into leukemia biology, and lead to the identification of novel microRNA - target gene interactions of potential relevance for acute myeloid leukemia treatment.

Clathrate hydrate formation after CO2-H2O vapour deposition.

We study vapour condensation of carbon dioxide and water at 77 K in a high-vacuum apparatus, transfer the sample to a piston-cylinder apparatus kept at 77 K and subsequently heat it at 20 MPa to 200 K. Samples are monitored by in situ volumetric experiments and after quench-recovery to 77 K and 1 bar by powder X-ray diffraction. At 77 K a heterogeneous mixture of amorphous solid water (ASW) and crystalline carbon dioxide is produced, both by co-deposition and sequential deposition of CO(2) and H(2)O. This heterogeneous mixture transforms to a mixture of cubic structure I carbon dioxide clathrate and crystalline carbon dioxide in the temperature range 160-200 K at 20 MPa. However, no crystalline ice is detected. This is, to the best of our knowledge, the first report of CO(2) clathrate hydrate formation from co-deposits of ASW and CO(2). The presence of external CO(2) vapour pressure in the annealing stage is not necessary for clathrate formation. The solid-solid transformation is accompanied by a density increase. Desorption of crystalline CO(2) atop the ASW sample is inhibited by applying 20 MPa in a piston-cylinder apparatus, and ultimately the clathrate is stabilized inside layers of crystalline CO(2) rather than in cubic or hexagonal ice. The vapour pressure of carbon dioxide needed for clathrate hydrate formation is lower by a few orders of magnitude compared to other known routes of CO(2) clathrate formation. The route described here is, thus, of relevance for understanding formation of CO(2) clathrate hydrates in astrophysical environments.

Cryoflotation: densities of amorphous and crystalline ices.

We present an experimental method aimed at measuring mass densities of solids at ambient pressure. The principle of the method is flotation in a mixture of liquid nitrogen and liquid argon, where the mixing ratio is varied until the solid hovers in the liquid mixture. The temperature of such mixtures is in the range of 77-87 K, and therefore, the main advantage of the method is the possibility of determining densities of solid samples, which are instable above 90 K. The accessible density range (~0.81-1.40 g cm(-3)) is perfectly suitable for the study of crystalline ice polymorphs and amorphous ices. As a benchmark, we here determine densities of crystalline polymorphs (ices I(h), I(c), II, IV, V, VI, IX, and XII) by flotation and compare them with crystallographic densities. The reproducibility of the method is about ±0.005 g cm(-3), and in general, the agreement with crystallographic densities is very good. Furthermore, we show measurements on a range of amorphous ice samples and correlate the density with the d spacing of the first broad halo peak in diffraction experiments. Finally, we discuss the influence of microstructure, in particular voids, on the density for the case of hyperquenched glassy water and cubic ice samples prepared by deposition of micrometer-sized liquid droplets.

HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems.

This article describes the design of HuCAL (human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100-300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.

How many amorphous ices are there?

Many acronyms are used in the literature for describing different kinds of amorphous ice, mainly because many different preparation routes and many different sample histories need to be distinguished. We here introduce these amorphous ices and discuss the question of how many of these forms are of relevance in the context of polyamorphism. We employ the criterion of reversible transitions between amorphous "states" in finite intervals of pressure and temperature to discriminate between independent metastable amorphous "states" and between "substates" of the same amorphous "state". We argue that the experimental evidence suggests we should consider there to be three polyamorphic "states" of ice, namely low-(LDA), high-(HDA) and very high-density amorphous ice (VHDA). In addition to the realization of reversible transitions between them, they differ in terms of their properties, e.g., compressibility, or number of "interstitial" water molecules. Thus they cannot be regarded as structurally relaxed variants of each other and so we suggest considering them as three distinct megabasins in an energy landscape visualization.

Pressure-amorphized cubic structure II clathrate hydrate: crystallization in slow motion.

A range of techniques has so far been employed for producing amorphous aqueous solutions. In case of aqueous tetrahydrofuran (THF) this comprises hyperquenching of liquid droplets, vapour co-deposition and pressure-induced amorphization of the crystalline cubic structure II clathrate. All of these samples are thermally labile and crystallize at temperatures above 110 K. We here outline a variant of the pressure-amorphization protocol developed by Suzuki [Phys. Rev. B, 2004, 70, 172108], which results in a highly crystallization resistant amorphous THF hydrate. The hydrate produced according to our protocol (annealing to 180 K at 1.8 GPa rather than to 150 K at 1.5 GPa) does not transform to the cubic structure II THF clathrate even at 150 K. We track the reason for this higher stability to the presence of crystalline remnants when following the Suzuki protocol, which are removed when using our protocol involving higher pressures and an annealing step. These crystalline remnants later serve as crystallization seeds lowering the thermal stability of the amorphous sample. Our protocol thus makes a purely amorphous THF hydrate available to the research community. We use powder X-ray diffraction to study the process of nucleation and slow crystal growth in the temperature range 160-200 K and find that the local cage structure and periodicity of the fully crystalline hydrate develops even at the earliest stages of crystallization, when the "clathrate crystal" has a size of about two unit cells.

Hexagonal ice transforms at high pressures and compression rates directly into "doubly metastable" ice phases.

We report compression and decompression experiments of hexagonal ice in a piston cylinder setup in the temperature range of 170-220 K up to pressures of 1.6 GPa. The main focus is on establishing the effect that an increase in compression rate up to 4000 MPa/min has on the phase changes incurred at high pressures. While at low compression rates, a phase change to stable ice II takes place (in agreement with earlier comprehensive studies), we find that at higher compression rates, increasing fractions and even pure ice III forms from hexagonal ice. We show that the critical compression rate, above which mainly the metastable ice III polymorph is produced, decreases by a factor of 30 when decreasing the temperature from 220 to 170 K. At the highest rate capable with our equipment, we even find formation of an ice V fraction in the mixture, which is metastable with respect to ice II and also metastable with respect to ice III. This indicates that at increasing compression rates, progressively more metastable phases of ice grow from hexagonal ice. Since ices II, III, and V differ very much in, e.g., strength and rheological properties, we have prepared solids of very different mechanical properties just by variation in compression rate. In addition, these metastable phases have stability regions in the phase diagrams only at much higher pressures and temperatures. Therefore, we anticipate that the method of isothermal compression at low temperatures and high compression rates is a tool for the academic and industrial polymorph search with great potential.

Raman spectroscopic study of the phase transition of amorphous to crystalline beta-carbonic acid.

What's the matter? The laboratory Raman spectra for carbonic acid (H(2)CO(3)), both for the beta-polymorph and its amorphous state, are required to detect carbonic acid on the surface of the pole caps of Mars in 2009, when the Mars Microbeam Raman Spectrometer lands on the planet. The picture shows a martian crater with ice of unknown composition, possibly containing carbonic acid (image adapted from DLR, with permission from ESA, DLR, and FU Berlin--G. Neukum).