X-ray Fluorescence Uptake Measurement of Functionalized Gold Nanoparticles in Tumor Cell Microsamples.

Quantitative cellular in vitro nanoparticle uptake measurements are possible with a large number of different techniques, however, all have their respective restrictions. Here, we demonstrate the application of synchrotron-based X-ray fluorescence imaging (XFI) on prostate tumor cells, which have internalized differently functionalized gold nanoparticles. Total nanoparticle uptake on the order of a few hundred picograms could be conveniently observed with microsamples consisting of only a few hundreds of cells. A comparison with mass spectroscopy quantification is provided, experimental results are both supported and sensitivity limits of this XFI approach extrapolated by Monte-Carlo simulations, yielding a minimum detectable nanoparticle mass of just 5 pg. This study demonstrates the high sensitivity level of XFI, allowing non-destructive uptake measurements with very small microsamples within just seconds of irradiation time.

Synthetic access to a hydrocarbon-soluble trifluorinated Ge(II) compound and its Sn(II) congener.

Trifluorinated germanium anions attracted attention of theoretical chemists already in the late 1990s to predict their physical and chemical properties. However these species were not synthesized in the laboratory, although substantial evidence for their existence was obtained from the mass spectrometry of GeF4. The present study shows that controlled fluorination of LMNMe2 (L = PhC(N(t)Bu)2, M = Ge, Sn) using HF·pyridine in toluene leads to the formation of [LH2](+)[MF3](-) under elimination of HNMe2. The products contain the trifluorinated Ge(II) and Sn(II) anionic species which are stabilized by interionic H···F bonds. The new compounds were characterized by single crystal X-ray structural analysis, NMR spectroscopy, and elemental analysis.

Structure determination from a single high-pressure-frozen virus crystal.

Successful cryogenic X-ray structure determination from a single high-pressure-frozen bovine enterovirus 2 crystal is reported. The presented high-pressure-freezing procedure is based on a commercially available device and allows the cryocooling of macromolecular crystals directly in their mother liquor without the time- and crystal-consuming search for optimal cryoconditions. The method is generally applicable and will allow cryogenic data collection from all types of macromolecular crystals.

The Effects of Napping on Wakefulness and Endurance Performance in Athletes: A Randomized Crossover Study.

Background: Athletes often experience poor sleep quality due to stress, altitude exposure, travel across different time zones, and pre-competition nervousness. Coaches use daytime naps to counteract the negative effects of fragmented nighttime sleep. Napping before competitions has also been used to enhance performance in athletes without sleep problems, with mixed results in previous studies, particularly for endurance performance. Thus, we investigated the effects of napping after partial sleep deprivation (PSD) on endurance performance and wakefulness in athletes. Methods: We recruited 12 healthy and trained participants (seven female and five male) for a randomized crossover study design. The participants underwent two test sessions: a five-hour night of sleep without a nap (noNap) and a five-hour night of sleep with a 30-min nap opportunity (Nap30). Participants recorded their sleep-wake rhythm one week before and during the study using the Consensus Sleep Diary-Core and the Morningness-Eveningness Questionnaire to examine their circadian rhythm type. We quantified PSD and the nap with pupillography (pupil unrest index, PUI), a subjective level of sleepiness questionnaire (Karolinska Sleepiness Scale, KSS), and polysomnography. After each night, participants performed a maximal cycling ergometry test to determine time to exhaustion (TTE) and maximal oxygen uptake (VO 2max). Results: Participants had an average sleep duration of 7.2 ± 0.7 h and were identified as moderately morning types (n = 5), neither type (n = 5), and moderately evening types (n = 2). There was a significant difference in both sleepiness parameters between the two conditions, with the PUI (p = 0.015) and KSS (p ≤ 0.01) significantly decreased at 5 h and nap compared with only 5 h of sleep. The PUI (p ≤ 0.01) and KSS (p ≤ 0.01) decreased significantly from before to after the nap. However, there was no significant difference in physical exercise test results between the conditions for TTE (p = 0.367) or VO 2max (p = 0.308). Conclusions: Our results suggest that napping after light PSD does not significantly influence endurance performance. We conclude that aerobic performance is a multidimensional construct, and napping after PSD may not enhance it. However, napping is an effective method to increase wakefulness and vigilance, which can be beneficial for sports competitions.

Fast high-pressure freezing of protein crystals in their mother liquor.

High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.

Hydrogen bonds as structural directive towards unusual polynuclear complexes: synthesis, structure, and magnetic properties of copper(II) and nickel(II) complexes with a 2-aminoglucose ligand.

The reaction of benzyl 2-amino-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside (HL) with the metal salts Cu(ClO(4))(2)6 H(2)O and Ni(NO(3))(2)6 H(2)O affords via self-assembly a tetranuclear mu(4)-hydroxido bridged copper(II) complex [(mu(4)-OH)Cu(4)(L)(4)(MeOH)(3)(H(2)O)](ClO(4))(3) (1) and a trinuclear alcoholate bridged nickel(II) complex [Ni(3)(L)(5)(HL)]NO(3) (2), respectively. Both complexes crystallize in the acentric space group P2(1). The X-ray crystal structure reveals the rare (mu(4)-OH)Cu(4)O(4) core for complex 1 which is mu(2)-alcoholate bridged. The copper(II) ions possess a distorted square-pyramidal geometry with an [NO(4)] donor set. The core is stabilized by hydrogen bonding between the coordinating amino group of the glucose backbone and the benzylidene protected oxygen atom O4 of a neighboring {Cu(L)} fragment as hydrogen-bond acceptor. For complex 2 an [N(4)O(2)] donor set is observed at the nickel(II) ions with a distorted octahedral geometry. The trinuclear isosceles Ni(3) core is bridged by mu(3)-alcoholate O3 oxygen atoms of two glucose ligands. The two short edges are capped by mu(2)-alcoholate O3 oxygen atoms of the two ligands coordinated at the nickel(II) ion at the vertex of these two edges. Along the elongated edge of the triangle a strong hydrogen bond (244 pm) between the O3 oxygen atoms of ligands coordinating at the two relevant nickel(II) ions is observed. The coordinating amino groups of the these two glucose ligands are involved in additional hydrogen bonds with O4 oxygen atoms of adjacent ligands further stabilizing the trinuclear core. The carbohydrate backbones in all cases adopt the stable (4)C(1) chair conformation and exhibit the rare chitosan-like trans-2,3-chelation. Temperature dependent magnetic measurements indicate an overall antiferromagnetic behavior for complex 1 with J(1)=-260 and J(2)=-205 cm(-1) (g=2.122). Compound 2 is the first ferromagnetically coupled trinuclear nickel(II) complex with J(A)=16.4 and J(B)=11.0 cm(-1) (g(1,2)=2.183, g(3)=2.247). For the high-spin nickel(II) centers a zero-field splitting of D(1,2)=3.7 cm(-1) and D(3)=1.8 cm(-1) is observed. The S=3 ground state of complex 2 is consistent with magnetization measurements at low temperatures.

(Z)-1,2:5,6-Di-O-isopropyl-idene-α-d-ribo-hexofuranos-3-ulose O-benzyl-oxime.

The title compound, C(19)H(25)NO(6), is a Z diastereomer in which the phenyl ring of the 3-benzyl-oxime substituent and the 5,6-O-isopropyl-idene acetal are both located on the Si-face of the C=N double bond. Inter-molecular C-H⋯O inter-actions result in helical chains along the b axis of the monoclinic unit cell.

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

Nickel(II) complexes with Schiff-base ligands derived from epimeric pyranose backbones as 2,3-chelators: modeling the coordination chemistry of chitosan.

Two mononuclear nickel(II) complexes with Schiff-base ligands derived from the epimeric sugars glucosamine and mannosamine have been synthesized. The X-ray crystal structure reveals a distorted octahedral geometry at the nickel(II) ions with an N4O2 donor set and the rare 2,3-chelation of the donor atoms of the carbohydrate backbone. Upon complexation only the glucopyranose ring maintains the 4C1 chair conformation, whereas the mannopyranose ring adopts the OS5 screw-boat conformation. Dimeric units of complex cations are formed by intermolecular hydrogen bonding which are further assembled by pi-stacking affording one-dimensional chains with a twofold screw symmetry.

Molecular electronic spin qubits from a spin-frustrated trinuclear copper complex.

The trinuclear copper(ii) complex [Cu3(saltag)(py)6]ClO4 (H5saltag = tris(2-hydroxybenzylidene)triaminoguanidine) was synthesized and characterized by experimental as well as theoretical methods. This complex exhibits a strong antiferromagnetic coupling (J = -298 cm-1) between the copper(ii) ions, mediated by the N-N diazine bridges of the tritopic ligand, leading to a spin-frustrated system. This compound shows a T2 coherence time of 340 ns in frozen pyridine solution, which extends to 591 ns by changing the solvent to pyridine-d5. Hence, the presented compound is a promising candidate as a building block for molecular spintronics.

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography.

Specific radiation damage can be used to determine phases de novo from macromolecular crystals. This method is known as radiation-damage-induced phasing (RIP). One limitation of the method is that the dose of individual data sets must be minimized, which in turn leads to data sets with low multiplicity. A solution to this problem is to use data from multiple crystals. However, the resulting signal can be degraded by a lack of isomorphism between crystals. Here, it is shown that serial synchrotron crystallography in combination with selective merging of data sets can be used to determine high-quality phases for insulin and thaumatin, and that the increased multiplicity can greatly enhance the success rate of the experiment.

Localising functionalised gold-nanoparticles in murine spinal cords by X-ray fluorescence imaging and background-reduction through spatial filtering for human-sized objects.

Accurate in vivo localisation of minimal amounts of functionalised gold-nanoparticles, enabling e.g. early-tumour diagnostics and pharmacokinetic tracking studies, requires a precision imaging system offering very high sensitivity, temporal and spatial resolution, large depth penetration, and arbitrarily long serial measurements. X-ray fluorescence imaging could offer such capabilities; however, its utilisation for human-sized scales is hampered by a high intrinsic background level. Here we measure and model this anisotropic background and present a spatial filtering scheme for background reduction enabling the localisation of nanoparticle-amounts as reported from small-animal tumour models. As a basic application study towards precision pharmacokinetics, we demonstrate specific localisation to sites of disease by adapting gold-nanoparticles with small targeting ligands in murine spinal cord injury models, at record sensitivity levels using sub-mm resolution. Both studies contribute to the future use of molecularly-targeted gold-nanoparticles as next-generation clinical diagnostic and pharmacokinetic tools.

X-ray focusing with efficient high-NA multilayer Laue lenses.

Multilayer Laue lenses are volume diffraction elements for the efficient focusing of X-rays. With a new manufacturing technique that we introduced, it is possible to fabricate lenses of sufficiently high numerical aperture (NA) to achieve focal spot sizes below 10 nm. The alternating layers of the materials that form the lens must span a broad range of thicknesses on the nanometer scale to achieve the necessary range of X-ray deflection angles required to achieve a high NA. This poses a challenge to both the accuracy of the deposition process and the control of the materials properties, which often vary with layer thickness. We introduced a new pair of materials-tungsten carbide and silicon carbide-to prepare layered structures with smooth and sharp interfaces and with no material phase transitions that hampered the manufacture of previous lenses. Using a pair of multilayer Laue lenses (MLLs) fabricated from this system, we achieved a two-dimensional focus of 8.4 × 6.8 nm2 at a photon energy of 16.3 keV with high diffraction efficiency and demonstrated scanning-based imaging of samples with a resolution well below 10 nm. The high NA also allowed projection holographic imaging with strong phase contrast over a large range of magnifications. An error analysis indicates the possibility of achieving 1 nm focusing.

Rapid cadmium SAD phasing at the standard wavelength (1†Å).

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Šis demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP-divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths (i.e. no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing.

Mix-and-diffuse serial synchrotron crystallography.

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.

Structural and Functional Characterization of Plasmodium falciparum Nicotinic Acid Mononucleotide Adenylyltransferase.

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)-in the product-bound state with NaAD and complexed with an α,ß-non-hydrolizable ATP analog-the structures were determined to a resolution of 2.2Å and 2.5Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences.

Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering.

Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D1/2 = 147.5 ±â€…19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures.

Native-like photosystem II superstructure at 2.44 Å resolution through detergent extraction from the protein crystal.

Photosystem II (PSII) catalyzes a key step in photosynthesis, the oxidation of water to oxygen. Excellent structural models exist for the dimeric PSII core complex of cyanobacteria, but higher order physiological assemblies readily dissociate when solubilized from the native thylakoid membrane with detergent. Here, we describe the crystallization of PSII from Thermosynechococcus elongatus with a postcrystallization treatment involving extraction of the detergent C12E8. This resulted in a transition from Type II to Type I-like membrane protein crystals and improved diffraction to 2.44 Å resolution. The obtained PSII packing in precise rows, interconnected by specific pairs of galactolipids and a loop in the PsbO subunit specific to cyanobacteria, is superimposable with previous electron microscopy images of the thylakoid membrane. The study provides a detailed model of such a superstructure and its organization of light-harvesting pigments with possible implications for the understanding of their efficient use of solar energy.

Room-temperature macromolecular serial crystallography using synchrotron radiation.

A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Šresolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

Chiral vanadium(V) complexes with 2-aminoglucose Schiff-base ligands and their solution configurations: synthesis, structures, and DFT calculations.

The sugar-modified Schiff-base ligands derived from benzyl 2-deoxy-2-salicylideneamino-α-D-glucopyranoside (H2L(5-Br) and H2L(3-OMe)) were used to prepare the chiral oxidovanadium(V) complexes [VO(L(5-Br))(OMe)] (1) and [VO(L(3-OMe))(OMe)] (2) which can be isolated from a methanol solution as the six-coordinate complexes with an additional methanol ligand [VO(L(5-Br))(OMe)(MeOH)] (1-MeOH) and [VO(L(3-OMe))(OMe) (MeOH)] (2-MeOH). Both complexes crystallize in the orthorhombic space group P2(1)2(1)2(1) together with two solvent molecules of methanol as 1-MeOH·2MeOH and 1-MeOH·2MeOH. In both crystal structures, only diastereomers with A configuration at the chiral vanadium centre (OC-6-24-A) are observed which corresponds to an cis configuration of the oxido group at the vanadium centre and the benzyl group at the anomeric carbon of the sugar backbone. Upon recrystallization of 2-MeOH from chloroform, the five-coordinate complex 2 was obtained which crystallizes in the monoclinic space group P2(1) with one co-crystallized chloroform molecule (2·CHCl3). For the chiral vanadium centre in 2·CHCl3, a C configuration (SPY-5-43-C) is observed which corresponds to an trans structure as far as the orientations of the oxido and benzyl groups are concerned. (1)H and (51)V NMR spectra of 1 and 2 indicate the presence of two diastereomers in solution. Their absolute configurations can be assigned based on the magnetic anisotropy effect of the oxidovanadium group. This effect leads to significant differences for the (1)H NMR chemical shifts of the H-2 (1.1 ppm) and H-3 protons (0.3 ppm) of the glucose backbone of the two diastereomers, with the downfield shift observed for the H-2 proton of the C-configured and the H-3 proton of the A-configured diastereomer at the vanadium centre. For 1 and 2 the difference between the (51)V NMR chemical shifts of the two diastereomers is 30 and 28 ppm, respectively. Also in the (13)C NMR significant chemical shift differences between the two diastereomers are observed for the carbon atoms C2 (2 ppm) and C3 (4 ppm). DFT calculations of the NMR chemical shift parameters have been performed which are in good agreement with the experimental data. Moreover, the isomerization mechanism between the diastereomers is analysed on the basis of DFT calculations which indicate the required presence of methanol molecules as protic donors.