The re-entrant transition from the molecular to atomic phases of dense fluids: The case of hydrogen.

A simple phenomenological thermodynamic model is developed to describe the chemical bonding and unbonding in homonuclear diatomic systems. This model describes the entire phase diagram of dimer-forming systems and shows a transition from monomers to dimers, with monomers favored at both very low and very high pressures, as well as at high temperatures. In the context of hydrogen, the former region corresponds to hydrogen present in most interstellar gas clouds, while the latter is associated with the long sought-after fluid metallic phase. The model predicts a molecular to atomic fluid transition in dense deuterium, which is in agreement with recently reported experimental measurements.

Effectiveness of bio-effectors on maize, wheat and tomato performance and phosphorus acquisition from greenhouse to field scales in Europe and Israel: a meta-analysis.

Biostimulants (Bio-effectors, BEs) comprise plant growth-promoting microorganisms and active natural substances that promote plant nutrient-acquisition, stress resilience, growth, crop quality and yield. Unfortunately, the effectiveness of BEs, particularly under field conditions, appears highly variable and poorly quantified. Using random model meta-analyses tools, we summarize the effects of 107 BE treatments on the performance of major crops, mainly conducted within the EU-funded project BIOFECTOR with a focus on phosphorus (P) nutrition, over five years. Our analyses comprised 94 controlled pot and 47 field experiments under different geoclimatic conditions, with variable stress levels across European countries and Israel. The results show an average growth/yield increase by 9.3% (n=945), with substantial differences between crops (tomato > maize > wheat) and growth conditions (controlled nursery + field (Seed germination and nursery under controlled conditions and young plants transplanted to the field) > controlled > field). Average crop growth responses were independent of BE type, P fertilizer type, soil pH and plant-available soil P (water-P, Olsen-P or Calcium acetate lactate-P). BE effectiveness profited from manure and other organic fertilizers, increasing soil pH and presence of abiotic stresses (cold, drought/heat or salinity). Systematic meta-studies based on published literature commonly face the inherent problem of publication bias where the most suspected form is the selective publication of statistically significant results. In this meta-analysis, however, the results obtained from all experiments within the project are included. Therefore, it is free of publication bias. In contrast to reviews of published literature, our unique study design is based on a common standardized protocol which applies to all experiments conducted within the project to reduce sources of variability. Based on data of crop growth, yield and P acquisition, we conclude that application of BEs can save fertilizer resources in the future, but the efficiency of BE application depends on cropping systems and environments.

From Atoms to Colloids: Does the Frenkel Line Exist in Discontinuous Potentials?

The Frenkel line has been proposed as a crossover in the fluid region of phase diagrams between a "nonrigid" and a "rigid" fluid. It is generally described as a crossover in the dynamical properties of a material and as such has been described theoretically using a very different set of markers from those with which is it investigated experimentally. In this study, we have performed extensive calculations using two simple yet fundamentally different model systems: hard spheres and square-well potentials. The former has only hardcore repulsion, while the latter also includes a simple model of attraction. We computed and analyzed a series of physical properties used previously in simulations and experimental measurements and discuss critically their correlations and validity as to being able to uniquely and coherently locate the Frenkel line in discontinuous potentials.

Krypton and the Fundamental Flaw of the Lennard-Jones Potential.

We have performed a series of neutron scattering experiments on supercritical krypton. Our data and analysis allow us to characterize the Frenkel line crossover in this model monatomic fluid. The data from our measurements was analyzed using Empirical Potential Structure Refinement to determine the short- and medium-range structure of the fluids. We find evidence for several shells of neighbors which form approximately concentric rings of density about each atom. The ratio of second to first shell radius is significantly larger than in any crystal structure. Modeling krypton using a Lennard-Jones potential is shown to give significant errors, notably that the liquid is overstructured. The true potential appears to be longer ranged and with a softer core than the 6-12 powerlaws permit.

How to determine solubility in binary mixtures from neutron scattering data: The case of methane and water.

It has recently been discovered that, when subjected to moderate amounts of pressure, methane dissolves in water to form binary mixtures of up to 40% molar methane. No significant solubility of water in methane is known. In these mixtures, the water hydrogen-bond network is largely complete and surrounds the methane molecules. The discovery of this dense mixture has once again highlighted the technical difficulties involved in accurately describing and sampling mixing phenomena both computationally and experimentally. Here, we present a systematic and critical study of the methods employed to characterize binary mixtures and their robustness. This study highlights the requirements needed to develop a quantitative understanding, and it proposes new and more accessible measures of miscibility to investigators, particularly for in silico analysis.

Frenkel Line in Nitrogen Terminates at the Triple Point.

Recent studies on supercritical nitrogen revealed clear changes in structural markers and dynamical properties when the coordination number approaches its maximum value. The line in P and T space where these changes occur is referred to as the Frenkel line. Here, we qualitatively reproduce such changes in the supercritical regime using the popular "optimized potential for liquid simulation" (OPLS) classical force field for molecular dynamics. Unfortunately, at 160 K, OPLS nitrogen predicts sublimation rather than producing a liquid phase; therefore, we developed our own force field to achieve quantitative agreement with experimental data. We confirm the asymptotic behavior of the coordination number on crossing the Frenkel line and note an associated change in the diffusion constant, consistent with the non-rigid to rigid liquid-like character of the "transition". The simulations allow us to track the Frenkel line to subcritical temperatures and demonstrate that it terminates at the triple point. This establishes the experimentally measurable changes, which could unequivocally determine the Frenkel line in other systems.

Structural Markers of the Frenkel Line in the Proximity of Widom Lines.

We have performed a neutron scattering experiment on supercritical fluid nitrogen at 160 K (1.27 TC) over a wide pressure range (7.8 MPa/0.260 g/mL-125 MPa/0.805 g/mL). This has enabled us to study the process by which nitrogen changes from a fluid that exhibits gaslike behavior to one that exhibits rigid liquidlike behavior at a temperature close to, but above, the critical temperature by crossing the Widom lines followed by the Frenkel line on pressure (density) increase. We find that the Frenkel line transition is indicated by a transition to a regime of rigid liquidlike behavior in which the coordination number remains constant within error, in agreement with our previous work at 300 K. The Frenkel line transition takes place at approximately the same density at 160 and 300 K. The data do not conclusively show an additional transition at the location of the known Widom lines. We find that behavior remains gaslike until the Frenkel line is crossed and our data support the hypothesis that Widom line transitions are density increase-driven.

Squeezing Oil into Water under Pressure: Inverting the Hydrophobic Effect.

The molecular structure of dense homogeneous fluid water-methane mixtures has been determined for the first time using high-pressure neutron-scattering techniques at 1.7 and 2.2 GPa. A mixed state with a fully H-bonded water network is revealed. The hydration shell of the methane molecules is, however, revealed to be pressure-dependent with an increase in the water coordination between 1.7 and 2.2 GPa. In parallel, ab initio molecular dynamics simulations have been performed to provide insight into the microscopic mechanisms associated with the phenomenon of mixing. These calculations reproduce the observed phase change from phase separation to mixing with increasing pressure. The calculations also reproduce the experimentally observed structural properties. Unexpectedly, the simulations show mixing is accompanied by a subtle enhancement of the polarization of methane. Our results highlight the key role played by fine electronic effects on miscibility and the need to readjust our fundamental understanding of hydrophobicity to account for these.

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ß-Lactam Production.

To produce high levels of ß-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for ß-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production.IMPORTANCEPenicillium rubens is an important industrial producer of ß-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

Pressure-Induced Miscibility Increase of CH4 in H2O: A Computational Study Using Classical Potentials.

Methane and water demix under normal (ambient) pressure and temperature conditions because of the polar nature of water and the apolar nature of methane. Recent experimental work has shown, though, that increasing the pressure to values between 1 and 2 GPa (10-20 kbar) leads to a marked increase of methane solubility in water, for temperatures which are well below the critical temperature for water. Here, we perform molecular dynamics simulations based on classical force fields-which are well-used and have been validated at ambient conditions-for different values of pressure and temperature. We find the expected increase in miscibility for mixtures of methane and supercritical water; however, our model fails to reproduce the experimentally observed increase in methane solubility at large pressures and below the critical temperature of water. This points to the need to develop more accurate force fields for methane and methane-water mixtures under pressure.

When immiscible becomes miscible-Methane in water at high pressures.

At low pressures, the solubility of gases in liquids is governed by Henry's law, which states that the saturated solubility of a gas in a liquid is proportional to the partial pressure of the gas. As the pressure increases, most gases depart from this ideal behavior in a sublinear fashion, leveling off at pressures in the 1- to 5-kbar (0.1 to 0.5 GPa) range with solubilities of less than 1 mole percent (mol %). This contrasts strikingly with the well-known marked increase in solubility of simple gases in water at high temperature associated with the critical point (647 K and 212 bar). The solubility of the smallest hydrocarbon, the simple gas methane, in water under a range of pressure and temperature is of widespread importance, because it is a paradigmatic hydrophobe and occurs widely in terrestrial and extraterrestrial geology. We report measurements up to 3.5 GPa of the pressure dependence of the solubility of methane in water at 100°C-well below the latter's critical temperature. Our results reveal a marked increase in solubility between 1 and 2 GPa, leading to a state above 2 GPa where the maximum solubility of methane in water exceeds 35 mol %.

Enzymatic network for production of ether amines from alcohols.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

Metabolism of ß-valine via a CoA-dependent ammonia lyase pathway.

Pseudomonas species strain SBV1 can rapidly grow on medium containing ß-valine as a sole nitrogen source. The tertiary amine feature of ß-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of ß-valine, a PsSBV1 gene library was prepared and used to complement the ß-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding ß-valinyl-coenzyme A ligase (BvaA) and two genes encoding ß-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate ß-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only ß-valine and to a lesser extent, 3-aminobutyrate and ß-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate ß-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Biochemical properties and crystal structure of a ß-phenylalanine aminotransferase from Variovorax paradoxus.

By selective enrichment, we isolated a bacterium that can use ß-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-ß-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The ß-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted ß-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-ß-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic ß-amino acids to yield (R)-ß-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the ß-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic ß-amino acid-converting aminotransferases may be identified.

Structural determinants of the ß-selectivity of a bacterial aminotransferase.

Chiral ß-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure ß-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-ß-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a ß-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A ß-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both ß- and α-amino acids.

Engineering of an enantioselective tyrosine aminomutase by mutation of a single active site residue in phenylalanine aminomutase.

By replacing a single active-site residue Cys107 with Ser in phenylalanine aminomutase (PAM), the enzyme gained tyrosine aminomutase (TAM) activity while retaining PAM activity and high enantioselectivity. This engineered enantioselective TAM also catalyzed formation of ß-tyrosine from p-coumaric acid and may prove to be useful for the synthesis of enantiopure ß-tyrosine and its derivatives.

[Serotypes of hepatitis C virus in mixed cryoglobulinaemia]. / Caratterizzazione sierotipica del virus dell'epatite C nella crioglobulinemia mista.

BACKGROUND: Several authors have studied the genotypic distribution of hepatitis C virus (HCV) in mixed cryoglobulinaemia (MC), which represents the most typical extra-hepatic manifestation of the HCV infection. On the other hand, at present no data are available on the HCV serotypic characterization of MC patients. METHODS: Thus, 28 serum specimens from HCV positive patients affected by MC (7 males and 21 females; mean age of 64 yrs, range 35-80) have been evaluated by a serotyping EIA method (Murex HCV Serotyping 1-6 Assay, Murex Diagnostics, Pomezia, Italy). RESULTS: The HCV serotype 1 was found in 13 patients (46.4%), serotype 2 in 5 patients (17.9%) and mixed serotypes in 3 patients (10.7%), showing, respectively, serotypes 1+2, 1+4, and 1+4+5. Moreover, the HCV serotype was not detectable in 7 patients (25.0%). CONCLUSIONS: These results, obtained by using a simple, serologic technique, agree with some literature data concerning MC patients from the same geographic region as ours (Venetian area). In addition, our findings appears to reflect merely the genotypic distribution of the HCV infection in the North Italy.