Microscopic Modeling of País Grape Seed Extract Absorption in the Small Intestine

The concentration profiles and the absorbed fraction (F) of the País grape seed extract in the human small intestine were obtained using a microscopic model simulation that accounts for the extracts'' dissolution and absorption. To apply this model, the physical and chemical parameters of the grape seed extract solubility (C_s), density (ρ), global mass transfer coefficient between the intestinal and blood content (k) (effective permeability), and diffusion coefficient (D) were experimentally evaluated. The diffusion coefficient (D = 3.45 × 10⁻⁶ ± 5 × 10⁻⁸ cm²/s) was approximately on the same order of magnitude as the coefficients of the relevant constituents. These results were chemically validated to discover that only the compounds with low molecular weights diffused across the membrane (mainly the (+)-catechin and (−)-epicatechin compounds). The model demonstrated that for the País grape seed extract, the dissolution process would proceed at a faster rate than the convective process. In addition, the absorbed fraction was elevated (F = 85.3%). The global mass transfer coefficient (k = 1.53 × 10⁻⁴ ± 5 × 10⁻⁶ cm/s) was a critical parameter in the absorption process, and minor changes drastically modified the prediction of the extract absorption. The simulation and experimental results show that the grape seed extract possesses the qualities of a potential phytodrug.KEY WORDS: dose absorption, mathematical modeling, País grape seed extract, simulation     

Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH^‡) and Eyring transition state entropies (ΔS^‡). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH_25°C = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH^‡ = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH_25°C = −20.5 ± 2.1 kcal/mol, ΔH^‡ = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl₂). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.     

Treatment with atorvastatin is associated with a better prognosis in chronic heart failure with systolic dysfunction: results from The Daunia Heart Failure Registry

Background

Few works have evaluated the effect of statins on left ventricular dysfunction in patients with chronic heart failure (CHF), by using tissue Doppler imaging (TDI). We therefore aimed to investigate whether atorvastatin treatment may influence prognosis and myocardial performance evaluated by TDI in subjects with CHF.

Methods

Five hundred thirty-two consecutive CHF outpatients enrolled in a local registry, the Daunia Heart Failure Registry, were prospectively analysed. 195 patients with CHF and left ventricular ejection fraction (LVEF) ≤40 %, either in treatment with atorvastatin (N: 114) or without statins (N: 81), underwent TDI examination. Adverse events were evaluated during follow-up.

Results

The atorvastatin group showed a lower incidence of adverse events (cardiac death: 0 % vs 7 %, p < 0.01), and better TDI performance (E/E’ 15 ± 5.7 vs 18 ± 8.3, p < 001) than controls. Ischaemic CHF patients in treatment with atorvastatin also showed a lower incidence of adverse events (death: 10 % vs 26 %, p < 0.05; sustained ventricular arrhythmias: 5 % vs 19 %, p < 0.05, cardiac death: 0 vs 8 %, p < 0.05) and better TDI performance (E/E’ ratio: 15.00 ± 5.68 vs 19.72 ± 9.14, p < 0.01; St: 353.70 ± 48.96 vs 303.33 ± 68.52 msec, p < 0.01) than controls. The association between atorvastatin and lower rates of cardiac death remained statistically significant even after correction in a multivariable analysis (RR 0.83, 95 % CI 0.71–0.96, p < 0.05 in CHF with LVEF ≤40 %; RR 0.77, 95 % CI 0.62–0.95, p < 0.05 in ischaemic CHF with LVEF ≤40 %).

Conclusions

Treatment with atorvastatin in outpatients with systolic CHF is associated with fewer cardiac deaths, and a better left ventricular performance, as assessed by TDI.     

A novel, functional and replicable risk gene region for alcohol dependence identified by genome-wide association study

Several genome-wide association studies (GWASs) reported tens of risk genes for alcohol dependence, but most of them have not been replicated or confirmed by functional studies. The present study used a GWAS to search for novel, functional and replicable risk gene regions for alcohol dependence. Associations of all top-ranked SNPs identified in a discovery sample of 681 African-American (AA) cases with alcohol dependence and 508 AA controls were retested in a primary replication sample of 1,409 European-American (EA) cases and 1,518 EA controls. The replicable associations were then subjected to secondary replication in a sample of 6,438 Australian family subjects. A functional expression quantitative trait locus (eQTL) analysis of these replicable risk SNPs was followed-up in order to explore their cis-acting regulatory effects on gene expression. We found that within a 90 Mb region around PHF3-PTP4A1 locus in AAs, a linkage disequilibrium (LD) block in PHF3-PTP4A1 formed the only peak associated with alcohol dependence at p<10⁻⁴. Within this block, 30 SNPs associated with alcohol dependence in AAs (1.6×10⁻⁵≤p≤0.050) were replicated in EAs (1.3×10⁻³≤p≤0.038), and 18 of them were also replicated in Australians (1.8×10⁻³≤p≤0.048). Most of these risk SNPs had strong cis-acting regulatory effects on PHF3-PTP4A1 mRNA expression across three HapMap samples. The distributions of −log(p) values for association and functional signals throughout this LD block were highly consistent across AAs, EAs, Australians and three HapMap samples. We conclude that the PHF3-PTP4A1 region appears to harbor a causal locus for alcohol dependence, and proteins encoded by PHF3 and/or PTP4A1 might play a functional role in the disorder.     

Effect of Acetate and Carbonate Buffers on the Photolysis of Riboflavin in Aqueous Solution: A Kinetic Study

The photolysis of riboflavin (RF) in the presence of acetate buffer (pH 3.8–5.6) and carbonate buffer (pH 9.2–10.8) has been studied using a multicomponent spectrophotometric method for the simultaneous assay of RF and its photoproducts. Acetate and carbonate buffers have been found to catalyze the photolysis reaction of RF. The apparent first-order rate constants for the acetate-catalyzed reaction range from 0.20 to 2.86 × 10⁻⁴ s⁻¹ and for the carbonate-catalyzed reaction from 3.33 to 15.89 × 10⁻⁴ s⁻¹. The second-order rate constants for the interaction of RF with the acetate and the carbonate ions range from 2.04 to 4.33 × 10⁻⁴ M⁻¹ s⁻¹ and from 3.71 to 11.80 × 10⁻⁴ M⁻¹ s⁻¹, respectively. The k-pH profile for the acetate-catalyzed reaction is bell shaped and for the carbonate-catalyzed reaction a steep curve. Both HCO₃⁻ and CO₃^2 − ions are involved in the catalysis of the photolysis reaction in alkaline solution. The rate constants for the HCO₃⁻ and CO₃^2 − ions catalyzed reactions are 0.72 and 1.38 × 10⁻³ M⁻¹ s⁻¹, respectively, indicating a major role of CO₃^2 − ions in the catalysis reaction. The loss of RF fluorescence in acetate buffer suggests an interaction between RF and acetate ions to promote the photolysis reaction. The optimum stability of RF solutions is observed in the pH range 5–6, which is suitable for pharmaceutical preparations.KEY WORDS: acetate effect, carbonate effect, photolysis, riboflavin, spectrophotometric assay     

Pathogenic and commensal Escherichia coli from irrigation water show potential in transmission of extended spectrum and AmpC β‐lactamases determinants to isolates from lettuce

There are few studies on the presence of extended-spectrum β-lactamases and AmpC β-lactamases (ESBL/AmpC) in bacteria that contaminate vegetables. The role of the production environment in ESBL/AmpC gene transmission is poorly understood. The occurrence of ESBL/AmpC in Escherichia coli (n = 46) from lettuce and irrigation water and the role of irrigation water in the transmission of resistant E. coli were studied. The presence of ESBL/AmpC, genetic similarity and phylogeny were typed using genotypic and phenotypic techniques. The frequency of β-lactamase gene transfer was studied in vitro. ESBLs/AmpC were detected in 35 isolates (76%). Fourteen isolates (30%) produced both ESBLs/AmpC. Prevalence was highest in E. coli from lettuce (90%). Twenty-two isolates (48%) were multi-resistant with between two and five ESBL/AmpC genes. The major ESBL determinant was the CTX-M type (34 isolates). DHA (33% of isolates) were the dominant AmpC β lactamases. There was a high conjugation efficiency among the isolates, ranging from 3.5 × 10⁻² to 1 × 10⁻² ± 1.4 × 10⁻¹ transconjugants per recipient. Water isolates showed a significantly higher conjugation frequency than those from lettuce. A high degree of genetic relatedness between E. coli from irrigation water and lettuce indicated possible common ancestry and pathway of transmission.     

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g⁻¹ protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g⁻¹ protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.Microorganisms play a crucial role in soil genesis by facilitating mineralization not only for soil organic matter (Leigh Mascarelli, 2009) but also for prevalent and persistent pollutants, such as polyaromatic hydrocarbons (PAH) and heterocyclic aromatic compounds (Lu et al., 2011). Such compounds are common additives in crude oil and industrial chemical products, such as dyes, flavouring compounds, plasticizers, perfumes, pesticides and insect repellent as well as, more recently, microelectronics, printed circuit boards, silk screen printing devices, optical disks and black colour tube matrices (Kästner, 2000; Chae et al., 2002). Multiple microorganisms can obtain energy from such hydrophobic aromatics (Lu et al., 2011), which are primarily released into the environment through anthropogenic activities. When bacteria are confronted with aromatic compounds, the cells encounter an interesting contradiction. On the one hand, such chemical species can be mineralized to yield carbon and energy for growth, which allows microorganisms to colonize niches that are refractory to other microbes (Dominguez-Cuevas et al., 2006; Lu et al., 2011). On the other hand, aromatic compounds over a certain threshold are toxic for bacteria because they partition and disorganize the cell membrane by removing lipids and proteins, which leads to cell death (Sikkema et al., 1995; von Wallbrunn et al., 2003). To cope with such activities, bacteria have developed multiple chemical tolerance mechanisms and an extensive enzyme arsenal (e.g. oxygenases, O-demetylases, CoA synthases and ligases, aldolases, alcohol dehydrogenases and α/β hydrolases) for mineralizing aromatic compounds by submitting them to the central metabolism (Pérez-Pantoja et al., 2008; 2009; Vilchez-Vargas et al., 2013).Recent studies suggest that multifunctional esterase/lipase-like proteins from the α/β hydrolase family that can hydrolyse both C-C and C-O bonds may exist in nature at much higher levels than previously thought (Alcaide et al., 2013). From an ecological perspective, such proteins may contribute to global carbon cycling processes for complex substrates, including recalcitrant organic pollutants. From a biotechnological perspective, such proteins may open unexpected research avenues for biotechnology applications. Esterases/lipases from the α/β hydrolase family have demonstrated such activities for catechol and biphenyl derivatives (Alcaide et al., 2013). However, the potential implication for the degradation of and biotechnology of complex organic molecules with two or more aromatic rings, specifically benzene rings (polyaromatic hydrocarbons – PAH), has not been described, even though the PAH degradation phenomenon is relatively well known (Pérez-Pantoja et al., 2008; 2009; Seo et al., 2009). Such activities are especially significant because the chemistry for PAH esters has been a subject of considerable interest due to their chemical and physical properties (e.g. polymers produced with versatile photo-reactivities) (Noh et al., 2001; Chae et al., 2002; Zhu et al., 2011). Heterogeneous catalysts have successfully been used to produce PAH-like (naphthalene, anthracene, phenanthrene, benzyl and phthalate) derivatives (Noh et al., 2001; Zhu et al., 2011; Maruyama et al., 2012). However, to the best of our knowledge, no study in the specialized literature has involved identification of potential applications for esterase/lipase-like proteins as catalysts for modifying PAHs.Because contaminated environments and microbial communities derived therefrom are an excellent source for enzymes that act on aromatics (Pérez-Pantoja et al., 2008; 2009; Guazzaroni et al., 2013), we now ask whether such α/β hydrolases can be identified therein. We generated a subset of 5500 clones (in Escherichia coli EPI300-T1^R; Epicentre Biotechnologies; Madison, WI, USA; Alcaide et al., 2013) from a naphthalene-enriched community (CN1) derived from PAH-contaminated soil (Guazzaroni et al., 2013), which included nearly 0.17 Gbp of community genomes, that were evaluated for their ability to hydrolyse α-naphthyl acetate (Reyes-Duarte et al., 2012). Four unique positive clones (hit rate 1:1375) were identified as active, and one (herein, CN1E1) was selected for in-depth analysis due to its high activity (halo/colour formation) and capacity for hydrolysis in agar-based assays using the model PAH ester naphthalene carboxylic acid methyl ester (Fig. 1). The insert [30 164 bp; nucleotide sequence available at the National Center for Biotechnology Information (NCBI) under accession number SRP030024] was sequenced [Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at LifeSequencing S.L., Valencia, Spain], analysed and compared with the sequences in the NCBI non-redundant public database (Altschul et al., 1997). Thirty-one predicted open reading frames were identified (Lukashin and Borodovsky, 1998); one encoded a putative esterase (CN1E1) with the α/β hydrolase fold. The protein [311 amino acids (AA); molecular weight (MW) 33 146 Da; isoelectric point (pI) 6.34] was produced in a soluble form (Fig. 2) upon expression in the pET-46 Ek/LIC vector (using the forward and reverse primers 5′-GACGACGACAAGATGGCGGTAGATCCG-3′ and 5′-GAGGAGAAGCCCGGTTATCTCGGTCCGGC-3′ respectively) and E. coli BL21 (DE3) (conditions described in Alcaide et al., 2013). The sequence was analysed, which indicated that it belonged to the α/β hydrolase superfamily; the esterase/lipase-like protein from Paenibacillus mucilaginosus 3016 ({"type":"entrez-protein","attrs":{"text":"YP_005313749.1","term_id":"379721618","term_text":"YP_005313749.1"}}YP_005313749.1) is the most similar enzyme (AA sequence identity: 54%; similarity: 68%). It was also homologous (≤ 56% identity) to predicted esterases/lipases from uncultured microorganisms derived from soil and marine sediment samples (Fig. 3). This enzyme can be categorized in the microbial family IV described by Arpigny and Jaeger (1999); it includes a typical -GlyxSerxGly-motif and presumptive Ser-Asp-His catalytic triad (Ser155, Asp250 and His280). CN1E1 was structurally most similar to a lipase/esterase-like protein from Alicyclobacillus acidocaldarius [identity: 45%; Protein Data Bank (PDB) code 2HM7_A].Open in a separate windowFigure 1Hydrolytic phenotype for E. coli BL21 (DE3) that either expressed or did not express CN1E1 using the pET-46 Ek/LIC vector. The cells were plated on fresh Luria Bertani (LB) plates with ampicillin (50 μg ml⁻¹). The plates [with 1.0 mM isopropyl-β-D-galactopyranoside (IPTG)] were incubated for 12 h at 37°C and then covered with a second layer that included the substrate [20 ml 5 mM N-(2-hydroxyethyl) piperazine-N′-(3-propanesulfonic acid) (EPPS) buffer, pH 8.0, 0.4% agarose and 320 μl of a naphthalene carboxylic acid methyl ester solution in acetonitrile (100 mg ml⁻¹)]. The active phenotype was apparent due to the colour change resulting from substrate hydrolysis and acetic acid formation, which changes the pH indicator from red to yellow.Open in a separate windowFigure 2A Coomassie-stained SDS-PAGE gel showed that the active form of the protein CN1E1 was over-expressed in E. coli at 16°C. The expression and purification conditions were reported in Martínez-Martínez and colleagues (2013). As shown, a high percentage of the protein was produced in its soluble form, which yielded a purity greater than 98% after a single His₆-tag purification step. Abbreviation: MW, molecular weight marker. The lanes include the following: lane 1, soluble cell fraction after induction with 1.0 mM IPTG; lane 2, soluble cell fraction without IPTG; lane 3, molecular weight marker (from top to bottom: 225, 150, 100, 75, 50, 35, 25 and 15 kDa); and lane 4, pure protein after His₆-tag purification.Open in a separate windowFigure 3The unrooted circular neighbour-joining tree indicates the polypeptide sequence phylogenetic positions for the CN1E1 enzyme (in boldface) and reference hydrolases. The tree was constructed using an aligned 297 AA-long sequence. The GenBank and PDB accession numbers are indicated in brackets. For the dendrogram construction details, see Martínez-Martínez and colleagues (2013) and Tamura and colleagues (2007). The Family V cluster includes the sequences with the following accession numbers: YP960710.1, {"type":"entrez-protein","attrs":{"text":"ADP98993.1","term_id":"311696120","term_text":"ADP98993.1"}}ADP98993.1, ZP01735705.1, ZP01735705.1, {"type":"entrez-protein","attrs":{"text":"CAE54381.1","term_id":"50871368","term_text":"CAE54381.1"}}CAE54381.1, ZP01307774.1, {"type":"entrez-nucleotide","attrs":{"text":"X53869","term_id":"44522","term_text":"X53869"}}X53869, {"type":"entrez-protein","attrs":{"text":"AEO74498.1","term_id":"347304384","term_text":"AEO74498.1"}}AEO74498.1, YP001347584.1, NP251639.1, YP790224.1, YP0029804424.1, YP002909304.2, YP001810250.1, ZP03570306.1, YP299126.1, YP583166.1, YP002005156.1, 725653.1 and YP006884923.1. The Family VI cluster includes YP007541785, WP007625024, WP008294645, S78600 and PDB 3CN7. The scale bar represents 0.2 substitutions per position. The lipase/esterase families are depicted based on the Arpigny and Jaeger (1999) classification system. The abbreviations are as follows: EST, esterase; LIP, lipase; HYP, hypothetical protein; ACTIN, Actinobacteria; FIR, Firmicutes; PLANCT, Planctomycetes; PROT, Proteobacteria; UNCB, uncultured bacterium.The pure enzyme was most active at 25–30°C and pH 8.5 (Fig. 4); it retained approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. The substrates used herein included 12 model esters [6 p-nitrophenol (pNP) and 6 triacylglycerol] and 86 structurally different esters (Martínez-Martínez et al., 2013). Based on the specific activities determined (units g⁻¹; one unit is the amount of enzyme that hydrolyses 1 μmol of substrate per min under the assay conditions, as reported by Martínez-Martínez et al., 2013), CN1E1 showed a capacity for accepting 61 esters with different alcohol and acid moieties (Fig. 5). Considering the acyl chain length and p-nitrophenyl ester hydrolysis, CN1E1 was most active with pNP-acetate (∼ 55 000 units g⁻¹ at 40°C and pH 8.0); it also hydrolysed pNP-dodecanoate, albeit at a rate of four orders of magnitude lower. As shown in Fig. 5, pNP esters were the preferred substrates among the examined esters. This enzyme hydrolysed short-chain triacylglycerols that ranged from triacetin (which was preferred; ∼ 23 000 units g⁻¹ at 40°C and pH 8.0) to tricaproin (∼ 21 units g⁻¹) and short-chain halogenated and non-halogenated alkyl and aryl esters; methyl bromoacetate (∼ 31 000 units g⁻¹) and butyl acetate (∼ 11 700 units g⁻¹) were preferred respectively (Fig. 5). Because the enzyme preferred short-chain triacylglycerols and short-to-medium size alkyl and aryl esters, the α/β-hydrolase is likely an esterase. It also showed a capacity for accepting tri-O-acetyl-glucal (∼ 12 500 units g⁻¹), the carbohydrate ester α-D-glucose pentaacetate (∼ 373 units g⁻¹) and hydroxycinnamic acid-like esters, such as 2,5-dihydroxycinnamic acid methyl ester (∼ 10 700 units g⁻¹), methyl cinnamate (∼ 28 units g⁻¹) and caffeic acid phenethyl ester (∼ 19 units g⁻¹) (Fig. 5). Such characteristics suggest that the enzyme can support polysaccharide degradation. Amino acid esters, such as L-proline and L-serine esters, were also hydrolysed (∼ 24–1.2 units g⁻¹); however, they were among the non-preferred substrates (Fig. 5). Under our assay conditions, the esterase was also active (from ∼ 609 to 5.2 units g⁻¹) and enantio-selective for (±)-menthyl-acetate, (±)-methyl lactate, (±)-neomenthyl-acetate, (±)-methyl-mandelate and (±)-glycidyl 4-nitrobenzoate with an (S)-preference (Fig. 5). γ-Butyrolactone (∼ 141 units g⁻¹), γ-valerolactone (∼ 81 units g⁻¹) and (±)-pantolactone [the (R) enantiomer was preferred; ∼ 606 units g⁻¹] were also accepted as substrates (Fig. 5).Open in a separate windowFigure 4The wild-type CN1E1 α/β hydrolase pH (A) and temperature (B) profiles. Esterase activity using pNP-propionate (at 410 nm) was determined as described previously (Martínez-Martínez et al., 2013). The standard deviation (SD) for the triplicate assays is shown. We examined pH values between 4.0 and 9.5 and temperatures between 4 and 80°C to determine the optimal parameters. The following buffers were examined at 40 mM: sodium citrate (pH 4.0–4.5), sodium acetate (pH 5.0–6.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5–6.0), piperazine-N,N′-bis(ethanesulfonic acid) (PIPES) (pH 6.0–7.0), 4-(2-hydroxyethylpiperazine-1-ethanesulfonic acid (HEPES) (pH 7.0–8.0), K/Na-phosphate (pH 7.5), Tris-HCl (pH 8.5) and glycine (pH 9.0–9.5). The pH was adjusted at 25°C. The pH and temperature profiles were collected at 40°C (panel A) and pH 7.0 (using 40 mM HEPES; panel B) respectively. In panels (A) and (B), 100% of the activity refers to 27.08 ± 1.80 and 21.17 ± 1.41 units mg⁻¹ respectively.Open in a separate windowFigure 5Substrate profile for the wild-type CN1E1 α/β hydrolase using a set of structurally diverse esters. The specific activities were calculated in triplicate as described by Martínez-Martínez and colleagues (2013) at 40°C in 20 mM HEPES buffer pH 7.0 (for pNP esters) or 5 mM EPPS buffer pH 8.0 (for the remaining esters). The standard deviation (SD) for the triplicate assays is shown. The chemicals used for the enzymatic experiments were the purest grade available and were purchased from Fluka-Aldrich-Sigma Chemical Co. (St. Louis, MO, USA). The hydroxycinnamic-like esters were supplied by Apin Chemicals (Oxon, UK), the methyl phenanthrene-3-carboxylate and methyl phenanthrene-9-carboxylate were supplied by Wuhan Farthest Chemical (Mainland, China), and the anthracene-9-carboxylic acid methyl ester and anthracene-3-carboxylic acid methyl ester were obtained from Alfa Aesar (Karlsruhe, Germany). Insets (A) to (C) in the figure represent a zoom for substrates hydrolysed at low rates.Because the active clone with CN1E1 actively hydrolysed naphthalene carboxylic acid methyl ester on an agar plate (Fig. 1), we further tested and quantified hydrolysis for the pure enzyme using this and other model PAH esters; the pH-indicator protocol (at 40°C and pH 8.0) used to examine hydrolysis for the above esters was also used for this experiment (Martínez-Martínez et al., 2013). CN1E1 showed broad reactivity towards PAH-like esters, including phthalate alkyl and aryl (benzene) esters (from ∼ 21 000 to 6100 units g⁻¹), alkyl esters of phenanthrene (from ∼ 20 500 to 7600 units g⁻¹), anthracene (from ∼ 18 400 to 11 000 units g⁻¹), naphthalene (from ∼ 10 000 to 7800 units g⁻¹) and protocatechuate (∼ 8800 units g⁻¹) and benzoate esters with substituent phenyl/benzene rings (from ∼ 7600 to 7200 units g⁻¹). The majority of such compounds were among the best substrates for CN1E1 (Fig. 5); this enzyme showed regio-selectivity for phenanthrene-3-carboxylate over methyl phenanthrene-9-carboxylate (2.7-fold) and, to a lesser extent, for anthracene-2-carboxylate over anthracene-9-carboxylate (1.7-fold). Although previous reports have shown that other esterases can degrade phthalate esters (Maruyama et al., 2005; Saito et al., 2010; Wu et al., 2013), to the best of our knowledge, the specialized literature has not reported an esterase with the capacity to degrade PAH esters with multiple aromatic (e.g. benzene) rings.The half-saturation (Michaelis) coefficient (K_m), catalytic rate constant (k_cat) and catalytic efficiency (k_cat/K_m) values were determined for the 14 best and structurally distinct substrates (2013) at pH 8.0 and 40°C (except for pNP-acetate were pH 7.0 was used to ensure substrate stability). The substrates analysed include pNP-acetate (the model p-nitrophenyl ester), triacetin (the model triacylglycerol), methyl bromoacetate (the model halogenated ester), tri-O-acetyl-glucal (the model polysaccharide-like ester), butyl acetate (the model alkyl ester), phenyl acetate (the model aryl ester), 2,5-dihydroxycinnamic acid methyl ester (the model hydroxycinnamic-like ester) and seven chemically distinct model PAH esters. As shown in 2013)

Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.KEY WORDS: ascorbic acid, cyanocobalamin, degradation, hydroxocobalamin, kinetics, two-component spectrometry     

Prevalence of Histidine Decarboxylase Genes of Gram-Positive Bacteria in Surströmming as Revealed by qPCR

Histamine is a degradation product of the bacterial decarboxylation of the amino acid histidine; such activity is determined by histidine decarboxylase encoded by a gene cluster, carried by some Gram-positive bacteria, that includes the hdcA gene. In this study, the presence of the hdcA gene in ready-to-eat surströmming samples collected from three producers based in Sweden was directly assessed via qPCR analysis for the very first time. Samples from producer A showed hdcA average gene abundance of 6.67 ± 0.13 Log cells/gene copies g⁻¹; in samples from producer B the average value attested at 5.56 ± 0.06 Log cells/gene copies g⁻¹, whereas for samples of producer C hdcA average gene abundance attested at 5.30 ± 0.08 Log cells/gene copies g⁻¹. ANOVA showed a significantly higher average hdcA gene copy number in samples from producer A, whereas no significant differences were seen between average values of hdcA gene copy numbers detected in samples from producer B and C. The hdcA gene copies detected in the present study could give an estimation of the load of potential histamine-producing bacteria in surströmming.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

CFH, VEGF, and PEDF genotypes and the response to intravitreous injection of bevacizumab for the treatment of age-related macular degeneration

We determined whether there is an association between complement factor H (CFH), high-temperature requirement A-1 (HTRA1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF) genotypes and the response to treatment with a single intravitreous injection of bevacizumab for age-related macular degeneration (AMD). Eighty-three patients with exudative AMD treated by bevacizumab injection were genotyped for three single nucleotide polymorphisms (SNPs; rs800292, rs1061170, rs1410996) in the CFH gene, a rs11200638-SNP in the HTRA1 gene, three SNPs (rs699947, rs1570360, rs2010963) in the VEGF gene, and four SNPs (rs12150053, rs12948385, rs9913583, rs1136287) in the PEDF gene using a TaqMan assay. The CT genotype (heterozygous) of CFH-rs1061170 was more frequently represented in nonresponders in vision than TT genotypes (nonrisk allele homozygous) at the time points of 1 and 3 months, while there was no CC genotype (risk allele homozygous) in our study cohort (p = 7.66 × 10⁻³, 7.83 × 10⁻³, respectively). VEGF-rs699947 was also associated with vision changes at 1 month and PEDF-rs1136287 at 3 months (p = 5.11 × 10⁻³, 2.05 × 10⁻², respectively). These variants may be utilized for genetic biomarkers to estimate visual outcomes in the response to intravitreal bevacizumab treatment for AMD.     

Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments

Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at μg liter⁻¹ levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 μg C liter⁻¹ in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10⁻² liter·μg⁻¹ of C·h⁻¹) ≫ maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10⁻² liter·μg⁻¹ of C·h⁻¹). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell⁻¹ level was achieved at ≤10 μg C liter⁻¹ than at >10 μg C liter⁻¹ with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at μg liter⁻¹ levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.     

Relative Bioavailability of Chlorothiazide from Mucoadhesive Compacts in Pigs

The relative bioavailability of chlorothiazide from mucoadhesive polymeric compacts is compared to commercial oral suspension in pigs. A single-dose randomized study was conducted in 12 healthy pigs that are 9–10 weeks old. After overnight fasting, pigs were divided into two groups of six animals. To the first group, a reference product containing 50 mg of chlorothiazide suspension, and in the second group, test product (mucoadhesive compacts) chlorothiazide (50 mg) was administered with 75 mL of water via gastric tubes. Blood samples were collected between 0 to 24 h using catheters inserted into the jugular vein. Plasma was separated by protein precipitation, and chlorothiazide concentrations were determined using a high-performance liquid chromatography method. The mean T_max and the C_max of chlorothiazide following the administration of oral suspension and mucoadhesive compacts were 0.58 ± 0.20 h and 682.97 ± 415.69 ng/mL and 2.17 ± 0.98 h and 99.42 ± 124.08 ng/mL, respectively. The K_el and T_1/2 of chlorothiazide were found to be 1.06 ± 0.28 h⁻¹ and 0.70 ± 0.21 h from suspension and 0.95 ± 1.11 h⁻¹ and 2.05 ± 1.90 h from the compacts, respectively. The T_max of mucoadhesive compacts were significantly longer (p < 0.05; 2.17 h) than the reference products (0.58 h), whereas the C_max of compacts were significantly lower (99 ng/mL) than the reference product (683 ng/mL; p < 0.05). The area under the curve (AUC) of compacts accounts only 50.15% (404.32 ± 449.93 ng h/mL) of the reference product’s AUC (806.27 ± 395.97 ng h/mL). The relative bioavailability of the compacts was lower than that of the suspension, and this may be due to the narrow window of absorption for chlorothiazide.Key words: bioavailability, chlorothiazide, mucoadhesive compacts, pigs     

Temporal changes of soil physic‐chemical properties at different soil depths during larch afforestation by multivariate analysis of covariance

Soil physic-chemical properties differ at different depths; however, differences in afforestation-induced temporal changes at different soil depths are seldom reported. By examining 19 parameters, the temporal changes and their interactions with soil depth in a large chronosequence dataset (159 plots; 636 profiles; 2544 samples) of larch plantations were checked by multivariate analysis of covariance (MANCOVA). No linear temporal changes were found in 9 parameters (N, K, N:P, available forms of N, P, K and ratios of N: available N, P: available P and K: available K), while marked linear changes were found in the rest 10 parameters. Four of them showed divergent temporal changes between surface and deep soils. At surface soils, changing rates were 262.1 g·kg⁻¹·year⁻¹ for SOM, 438.9 mg·g⁻¹·year⁻¹ for C:P, 5.3 mg·g⁻¹·year⁻¹ for C:K, and −3.23 mg·cm⁻³·year⁻¹ for bulk density, while contrary tendencies were found in deeper soils. These divergences resulted in much moderated or no changes in the overall 80-cm soil profile. The other six parameters showed significant temporal changes for overall 0–80-cm soil profile (P: −4.10 mg·kg⁻¹·year⁻¹; pH: −0.0061 unit·year⁻¹; C:N: 167.1 mg·g⁻¹·year⁻¹; K:P: 371.5 mg·g⁻¹ year⁻¹; N:K: −0.242 mg·g⁻¹·year⁻¹; EC: 0.169 μS·cm⁻¹·year⁻¹), but without significant differences at different soil depths (P > 0.05). Our findings highlight the importance of deep soils in studying physic-chemical changes of soil properties, and the temporal changes occurred in both surface and deep soils should be fully considered for forest management and soil nutrient balance.     

Characterization of the major histocompatibility complex locus association with Beh?et’s disease in Iran

IntroductionThe aim of this study was to characterize the association of human leukocyte antigen (HLA) B alleles and major histocompatibility complex (MHC) single nucleotide polymorphisms (SNPs) with Behçet’s disease (BD) in an Iranian dataset.MethodsThe association of three SNPs in the MHC region previously identified as the most associated in high-density genotyping studies was tested in a case–control study on 973 BD patients and 825 controls from Iran, and the association of HLA-B alleles was tested in a subset of 681 patients and 414 controls.ResultsWe found that HLA-B*51 (P = 4.11 × 10⁻⁴¹, OR [95% CI] = 4.63[3.66-5.85]) and B*15 confer risk for BD (P = 2.83 × 10⁻², OR [95% CI] = 1.75[1.08-2.84]) in Iranian, and in B*51 negative individuals, only the B*15 allele is significantly associated with BD (P = 2.51 × 10⁻³, OR [95% CI] = 2.40[1.37-4.20]). rs76546355, formerly known as rs116799036, located between HLA-B and MICA (MHC class I polypeptide-related sequence A), demonstrated the same level of association with BD as HLA-B*51 (P_adj = 1.78 × 10⁻⁴⁶, OR [95% CI] = 5.46[4.21-7.09], and P_adj = 8.34 × 10⁻⁴⁸, OR [95% CI] = 5.44[4.20-7.05], respectively) in the HLA-B allelotyped subset, while rs2848713 was less associated (P_adj = 7.14 × 10⁻³⁵, OR [95% CI] = 3.73[2.97-4.69]) and rs9260997 was not associated (P_adj = 1.00 × 10⁻¹). Additionally, we found that B*51 genotype-phenotype correlations do not survive Bonferroni correction, while carriers of the rs76546355 risk allele predominate in BD cases with genital ulcers, positive pathergy test and positive BD family history (2.31 × 10⁻⁴ ≤ P ≤ 1.59 × 10⁻³).ConclusionsWe found that the HLA-B*51 allele and the rs76546355/rs116799036 MHC SNP are independent genetic risk factors for BD in Iranian, and that positivity for the rs76546355/rs116799036 risk allele, but not for B*51, does correlate with specific demographic characteristics or clinical manifestations in BD patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0585-6) contains supplementary material, which is available to authorized users.     

Increased In Situ Intestinal Absorption of Phytoestrogenic Diarylheptanoids from Curcuma comosa in Nanoemulsions

Curcuma comosa has long been used as a gynecological medicine. Several diarylheptanoids have been purified from this plant, and their pharmacological effects were proven. However, there is no information about the absorption of C. comosa components to support the formulation usage. In the present study, C. comosa hexane extract and the mixture of its two major compounds, (4E,6E)-1,7-diphenylhepta-4,6-dien-3-ol (DA1) and (6E)-1,7-diphenylhept-6-en-3-ol (DA2), were formulated into nanoemulsions. The physical properties of the nanoemulsions and the in situ intestinal absorptions of DA1 and DA2 were evaluated. The results demonstrated the mean particle sizes at 0.207 ± 0.001 and 0.408 ± 0.014 μm, and the zeta potential at −14.57 ± 0.85 and −10.47 ± 0.32 mV for C. comosa nanoemulsion (C.c-Nano) and mixture of diarlylheptanoid nanoemulsions (DA-Nano), respectively. The entrapments of DA1 and DA2 were 76.61% and 75.41%, and 71.91% and 71.63% for C.c-Nano and DA-Nano, respectively. The drug loading ratios of DA1 and DA2 were 351.47 and 614.53 μg/mg, and 59.48 and 126.72 μg/mg for C.c-Nano and DA-Nano. The intestinal absorption rates of DA1 and DA2 were 0.329 ± 0.015 and 0.519 ± 0.026 μg/min/cm² in C.c-Nano, and 0.380 ± 0.006 and 0.428 ± 0.036 μg/min/cm² in DA-Nano, which were five to ten times faster than those in oil. In conclusion, the formulation in nanoemulsion forms obviously increased the intestinal absorption rate of diarylheptanoids.KEY WORDS: Curcuma comosa, diarylheptanoids, intestinal absorption, nanoemulsion, phytoestrogen     

Photodegradation and Stabilization of Betamethasone-17 Valerate in Aqueous/Organic Solvents and Topical Formulations

The effects of solvent [acetonitrile, methanol, and acetonitrile/water mixture (20:80, v/v)], buffer concentration (phosphate buffer, pH 7.5), ionic strength and commonly employed adjuvants on the photodegradation of betamethasone-17 valerate in cream and gel formulations have been studied on exposure to UV light (300–400 nm). A validated high-performance liquid chromatography method has been used to determine the parent compound and its photodegraded products. The photodegradation data in the studied solvents showed greater decomposition of the drug in solvents with a lower dielectric constant. A comparatively higher rate of photodegradation was observed in the cream formulation compared to that for the gel formulation. The kinetic treatment of the photodegradation data revealed that the degradation of the drug follows first-order kinetics and the apparent first-order rate constants for the photodegradation reactions, in the media studied, range from 1.62 to 11.30 × 10⁻³ min⁻¹. The values of the rate constants decrease with increasing phosphate concentration and ionic strength which could be due to the deactivation of the excited state and radical quenching. The second-order rate constant (k′) for the phosphate ion-inhibited reactions at pH 7.5 has been found to be 5.22 × 10⁻² M⁻¹ s⁻¹. An effective photostabilization of the drug has been achieved in cream and gel formulations with titanium dioxide (33.5–42.5%), vanillin (21.6–28.7%), and butyl hydroxytoluene (18.2–21.6%).Key words: betamethasone-17 valerate, creams and gels, kinetics, photodegradation, photostabilization     

Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number

Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of high-resolution structures, hydrodynamic radius and diffusion coefficient of evolutionary conserved RNA single strands (ssRNA) were calculated. The diffusion coefficients D of 17–174 nucleotides (nt) containing ssRNA depended on the number of nucleotides N with D = 4.56 × 10⁻¹⁰ N^−0.39 m² s⁻¹. The hydrodynamic radius R_H depended on N with R_H = 5.00 × 10⁻¹⁰ N^0.38 m. An average ratio of the radius of gyration and the hydrodynamic radius of 0.98 ± 0.08 was calculated in solution. The empirical law was tested by in solution measured hydrodynamic radii and radii of gyration and was found to be highly consistent with experimental data of evolutionary conserved ssRNA. Furthermore, the hydrodynamic behaviour of several evolutionary unevolved ribonucleic acids could be predicted. Based on atom-level shell-modelling of high-resolution structures and experimental hydrodynamic data, empirical models are proposed, which enable to predict the translational diffusion coefficient and molecular size of short RNA single strands solely on the basis of the polymer size.     

Development of bioelectrocatalytic activity stimulates mixed‐culture reduction of glycerol in a bioelectrochemical system

In a microbial bioelectrochemical system (BES), organic substrate such as glycerol can be reductively converted to 1,3-propanediol (1,3-PDO) by a mixed population biofilm growing on the cathode. Here, we show that 1,3-PDO yields positively correlated to the electrons supplied, increasing from 0.27 ± 0.13 to 0.57 ± 0.09 mol PDO mol⁻¹ glycerol when the cathodic current switched from 1 A m⁻² to 10 A m⁻². Electrochemical measurements with linear sweep voltammetry (LSV) demonstrated that the biofilm was bioelectrocatalytically active and that the cathodic current was greatly enhanced only in the presence of both biofilm and glycerol, with an onset potential of −0.46 V. This indicates that glycerol or its degradation products effectively served as cathodic electron acceptor. During long-term operation (> 150 days), however, the yield decreased gradually to 0.13 ± 0.02 mol PDO mol⁻¹ glycerol, and the current–product correlation disappeared. The onset potentials for cathodic current decreased to −0.58 V in the LSV tests at this stage, irrespective of the presence or absence of glycerol, with electrons from the cathode almost exclusively used for hydrogen evolution (accounted for 99.9% and 89.5% of the electrons transferred at glycerol and glycerol-free conditions respectively). Community analysis evidenced a decreasing relative abundance of Citrobacter in the biofilm, indicating a community succession leading to cathode independent processes relative to the glycerol. It is thus shown here that in processes where substrate conversion can occur independently of the electrode, electroactive microorganisms can be outcompeted and effectively disconnected from the substrate.