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Byline: Christiane Hartog (1), Konrad Reinhart (1) Keywords: Colloids; Crystalloids; Hydroxyethyl starch; Efficacy; Safety; Critically ill Abstract: Purpose To describe the risk--benefit profile of hydroxyethyl starch (HES). Methods Narrative review. Results (1) Efficacy: no single clinical study or systemic review has shown that administration of any HES solution confers a clinically relevant benefit compared to crystalloids in critically ill patients or surgical patients in need of volume replacement. Contrary to beliefs expecting a ratio of 4:1 or more for crystalloid to colloid volume need, recent studies of goal-directed resuscitation observed much lower ratios of between 1 and 1.6. (2) Safety: HES administration is associated with coagulopathy, nephrotoxicity, pruritus and increased long-term mortality. Clinical studies claiming that modern HES 130/0.4 is safe have serious methodological drawbacks and do not adequately address the safety concerns. Conclusions Given the complete lack of superiority in clinical utility studies and the wide spectrum of severe side effects, the use of HES in the ICU should be stopped. The belief that four times as much crystalloid as colloid fluid volume is needed for successful resuscitation is being seriously questioned. Author Affiliation: (1) Department of Anaesthesiology and Intensive Care Medicine, Friedrich Schiller University, Erlanger Allee 101, 07747, Jena, Germany Article History: Registration Date: 28/05/2009 Received Date: 15/12/2008 Accepted Date: 13/04/2009 Online Date: 17/06/2009 Article note: C. Hartog and K. Reinhart contributed equally to this work. The article arguing for this proposition is available at: doi: 10.1007/s00134-009-1520-6.

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coil engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobactersp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria. insect-microbe interactions | microbe-microbe interactions | synergy | Lepidoptera | pore-forming toxin

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x [10.sup.7] Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program RADDOSE after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half ([D.sub.1/2]). From these measurements, a dose limit of [D.sub.1/2] = 4.3 ([+ or -]0.3) x [10.sup.7] Gy was obtained. However, by considering other data quality indicators, an intensity reduction to IIn2 = In2 x [I.sub.0], corresponding to an absorbed dose of 3.0 x [10.sup.7] Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments. experimental dose limit | macromolecular cryocrystallography | radiation damage

It is generally assumed that crystallization begins with a small, crystalline nucleus. For proteins this paradigm may not be valid. Our numerical simulations show that under conditions typically used to produce protein crystals, small clusters of model proteins (particles with short-range, attractive interactions) cannot maintain a crystalline structure. Protein crystal nucleation is therefore an indirect, two-step process. A nucleus first forms and grows as a disordered, liquid-like aggregate. Once the aggregate grows beyond a critical size (about a few hundred particles) crystal nucleation becomes possible.

A novel technique was used to study the nucleation of crystal of a model protein from aqueous solutions.

The establishment of a quantitative connection between backbone-backbone hydrogen-bond (H-bond) length seen in protein crystal structure and (super 3h)J(sub NC') couplings across such bonds is limited by the coordinate precision of the X-ray structure. Very high-resolution X-structures are available for an immunoglobulin binding domain of streptococcal protein G. Findings showed that over the small range of N-O H-bond lengths for which (super 3h)J(sub NC') couplings are observable, the 32 measured (super 3h)J(sub NC') values can be fit to: (super 3h)J(sub NC') = -59000 exp(-4R(sub NO)) +/- 0.09 Hz, or R(sub NO) = 2.75 - 0.25 ln(-(super 3h)J(sub NC')) +/- 0.06 angstrom.

The reordering of radiation-damaged protein crystals is reported, under conditions of transport initiated by stress. The distorted crystal recrystallized after a certain density of radiation-induced defects are accumulated. Stress is caused by the defects at temperatures low enough to suppress diffusive mass transport, indicating that the defect induced reordering may be a general phenomenon.

The phase diagram of a protein-water system was measured using a model with only two parameters that characterize the interaction between protein molecules in the liquid and in the solid phase. The resulting phase diagram was then used to analyze the optimal conditions for the growth of protein crystals. Results suggested that the preferred mechanism for the growth of protein crystals is one with liquid-liquid phase separation as a first step.

Crystalline inorganic substances such as zeolites and pillared layer solids are extensively used in separation and catalysis operations. Cross linked protein crystals have been shown to be useful and remarkably stable catalysts for the successful derivation of useful microporous organic solids using the analytic method of macromolecular porosimetry. The procedure, its application and the results are analyzed and discussed.

We have developed methods for obtaining and characterizing three-dimensional maps of the reciprocal-space distribution of diffuse x-ray scattering from protein crystals, and have used the methods to study the nature of disorder in crystals of Staphylococcal nuclease. Experimentally obtained maps are 99.5% complete in the reciprocal-space resolution range of 10 [Angstrom]-2.5 [Angstrom], show symmetry consistent with the P[4.sub.1] space group of the unit cell, and are highly reproducible. Quantitative comparisons of the data with three-dimensional simulations imply liquid-like motions of the protein [Caspar, D. L. D., Clarage, J., Salunke, D. M. & Clarage, M. (1988) Nature (London) 332, 659-662], with a correlation length of 10 [Angstrom] and a root-mean-square displacement of 0.36 [Angstrom].

The crystalline structure of photosystem II is characterized by dimers of 17- and 11.4-nanometer units composed of polypeptides such as D1 and D2, cytochrome b559 and chlorophyll-binding polypeptides. This dimeric structure was observed from cryoelectron microscopy and projection mapping of the of the photosystem. The reaction center responsible for transforming water into oxygen can be found near the central cleft where polypeptides are most dense.

The crystal structure of human thymidylate synthase (TS) shows that its folding is mostly similar to that of the bacterial TS. The only major difference lies in the active site loop. The folding of the loop of human TS warrants a realignment when it binds the substrate, because the catalytic cysteine residue lies buried in the loop. This realignment mechanism makes the active site loop rotate by 180 degrees while binding the substrate. A drug resistance mutant TS, Y6H, brings in a four-fold reduction in fluoro-2'-deoxyuridylate affinity.

The different ways by which Bacillus thuringiensis accumulates large amounts of insecticidal protein crystals, used as biopesticides, are examined. B. thuringiensis creates protease-resistant inclusion bodies which are made easily digestible in the insect stomach, by several factors, among which are the secondary structure of the Cry proteins, the energy of the disulfide bonds, and the presence of additional chemicals.

A combination of confocal, fluorescence and differential interference contrast microscopy is used to study the effect of tethered streptavidin protein crystals on the bilayer curvature of several giant lipid unilamellar vesicles (GUVs). The analysis of the mechanism for the protein-mediated stabilization of cellular membranes prove that the crystalline streptavidin domains have a strong stabilizing effect on the lipid membranes, leading to a better and facilitated membrane budding.

The experimental phase diagram of urate oxidase in the case of poly ethylene glycol (PEG) is characterized by determining the solubility curve and the dilute part of the liquid-liquid phase separation. The study shows that urate oxidase is a good model to study protein/PEG mixtures in the general context of protein crystallization.

The mechanism of double substrate recognition of Escherichia coli branched-chain amino acid transferase is distinct from that exhibited by the aromatic amino acid and histidinol-phosphate aminotransferases. The crystal structure of the former with its natural substrate, glutamate, exhibits a ketimine spectra, whereas with the acidic substrate, glutarate, it is a Michaelis complex.

The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis. In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll. The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP. Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch.) tepidum, at 2.2-[Angstrom] and 1.5-[Angstrom] resolution, respectively. Tch. tepidum can grow at the highest temperature of all known purple bacteria, and the Tch. tepidum RC shows some degree of stability to high temperature. Comparison with the RCs of mesophiles, such as Blastochloris viridis, has shown that the Tch. tepidum RC possesses more Arg residues at the membrane surface, which might contribute to the stability of this membrane protein. The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair.

The capability to enhance or suppress the nucleation of protein crystals opens opportunities in various fundamental and applied areas, including protein crystallography, production of protein crystalline pharmaceuticals, protein separation, and treatment of protein condensation diseases. Herein, we show that the rate of homogeneous nucleation of lysozyme crystals passes through a maximum in the vicinity of the liquid-liquid phase boundary hidden below the liquidus (solubility) line in the phase diagram of the protein solution. We found that glycerol and polyethylene glycol (which do not specifically bind to proteins) shift this phase boundary and significantly suppress or enhance the crystal nucleation rates, although no simple correlation exists between the action of polyethylene glycol on the phase diagram and the nucleation kinetics. The control mechanism does not require changes in the protein concentration, acidity, and ionicity of the solution. The effects of the two additives on the phase diagram strongly depend on their concentration, which provides opportunities for further tuning of nucleation rates.

A microfludic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization trial is described. The study shows that the system can be used in individual research labs to answer fundamental questions in crystallization and can be used for crystallization of new targets.

Research that explains some morphological and kinetics phenomena that arise during layer-wise growth of protein crystals is presented. The possible implications of the unsteady dynamics of layer growth are considered.

The helical crystallization method developed by E.M. Wilson-Kubalek, R.E. Brown, H. Celia and R.A. Milligan is an improvement over the lipid layer crystallization technique. It has allowed structure determination from helical crystals more available to a wide array of systems. In addition, the formation of helical crystals is facilitated by displaying tubular ligands on a cylindrical template.

A Heavenly Advantage Researchers have long suspected that superior protein crystals can be grown in space, but they have never been able to put a number on the improvement. The [...], At last month's Sixth International Conference on Crystallization of Biological Macromolecules in Hiroshima, Japan, researchers compared notes on one of the biggest challenges in determining protein structure: growing the high-quality crystals needed for x-ray analysis. Coaxing such crystals to form is still an art, but the meeting saw the beginnings of a more systematic approach. One presentation quantified the advantage of growing crystals in space; another presented techniques for growing better crystals on Earth.

New experiments aboard the Russian space station Mir indicate that the microgravity of space may be useful for growing protein crystals. Some critics have asserted that protein crystal research in space is touted merely to provide a pretext for expensive projects such as Mir and the US space station Freedom. However, the experiments aboard Mir attained a 24% success rate that warrants further attempts to perfect the technique. Mir proved to be a more dependable vehicle for crystal research than the US space shuttle.

Issues concerning the calculations of the Gibbs free energy and the concentration profile of the interface between an aqueous solution and a protein crystal are reported.

Russian crystallographers were the first to crystallize protein through ultracentrifugation. Researcher V. Barynin announced in Jul 1987 that beef liver catalase and fungal catalase from Penicillium vitale had been synthesized using ultracentrifugation. However, the rebuttal view holds that a later method of ultracentrifugation differs significantly from the Russians' technique. For instance, the later method, which produced aspartic proteinase crystals from Trichoderma reesei, took 2 and 1/2 hours rather than 180 hours.