Characterization of a Family GH5 Xylanase with Activity on Neutral Oligosaccharides and Evaluation as a Pulp Bleaching Aid

A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.The catabolic breakdown of xylan is a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research as a renewable energy resource as well as for bioconversion of plant biomass into high-added-value products (21, 29, 37, 40). Biodegradation of xylan is a complex process that requires the coordinate action of several enzymes, among which xylanases (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8), cleaving internal linkages on the β-1,4-xylose backbone, play a key role.Most known xylanases are grouped into glycoside hydrolase (GH) families 10 and 11 (CAZy [Carbohydrate-Active enZYmes] database) (17), although a few xylanases have recently been ascribed to glycoside hydrolase families 5, 7, 8, and 43 (8, 9, 24). Among xylanases not grouped in the typical families GH10 and GH11, only two xylanases belonging to family GH5 have been biochemically characterized in detail. The enzymes XynA from Erwinia chrysanthemi (18, 39) and XynC from Bacillus subtilis (32) hydrolyze glucuronoxylan to branched xylooligosaccharides. The activity of Erwinia chrysanthemi XynA has also been evaluated on other substrates containing xylose, showing an absolute requirement for methyl-glucuronic substitutions. In this way, only methyl-glucuronic acid-branched oligosaccharides can be cleaved by XynA, whereas linear xylooligosaccharides or arabinoxylans are not cleaved by this enzyme (39). This type of xylanases must play an important role in complementing the action of GH10 and GH11 enzymes during depolymerization of glucuronoxylans in lignocellulosic fibers.Xylanases are widely used in the pulp industry to enhance the effectiveness of bleaching agents, thereby reducing the generation of toxic wastes (adsorbable organic halogens; AOX) (1, 38). Several reports have evaluated the effectiveness of family GH10 and GH11 xylanases on the bleaching process, showing that GH11 xylanases usually display better performance (7, 12), although there are many other factors that contribute to the bleach-boosting effect of a xylanase, such as the source of the pulp and the pulping process itself (6, 11). Besides their contribution to the increase in brightness, an innovative aspect of the application of xylanases is their contribution to the removal of hexenuronic acids (HexA) produced during the kraft cooking process, which can accelerate the brightness reversion (yellowing tendency) of paper (35). However, it remains to be known if all xylanases are capable of removing HexAs and/or enhancing bleachability.Bacillus sp. strain BP-7 is a xylanolytic strain isolated from agricultural soils (25). It shows a multiple enzymatic system for xylan degradation, including a GH11 xylanase cloned and characterized previously (13). In this work, we describe the identification and cloning of a second xylanase from the strain, belonging to the GH5 family. The enzyme hydrolyzes linear xylooligosaccharides, clearly differing from the two GH5 xylanases characterized up to date. The new enzyme has been tested on Eucalyptus pulps, showing good performance as a bleaching aid. The results obtained suggest an important role for the enzyme in xylan degradation and indicate the potential of this xylanase for biotechnological applications in the bioconversion of glucuronoxylan-containing biomass.     

Overview of the structure of all-AT oligonucleotides: organization in helices and packing interactions

We present the crystalline organization of 33 all-AT deoxyoligonucleotide duplexes, studied by x-ray diffraction. Most of them have very similar structures, with Watson-Crick basepairs and a standard average twist close to 36 degrees. The molecules are organized as parallel columns of stacked duplexes in a helical arrangement. Such organization of duplexes is very regular and repetitive: all sequences show the same pattern. It is mainly determined by the stacking of the terminal basepairs, so that the twist in the virtual TA base step between neighbor duplexes is always negative, approximately -22 degrees. The distance between the axes of parallel columns is practically identical in all cases, approximately 26 A. Interestingly, it coincides with that found in DNA viruses and fibers in their hexagonal phase. It appears to be a characteristic distance for ordered parallel DNA molecules. This feature is due to the absence of short range intermolecular forces, which are usually due to the presence of CG basepairs at the end of the oligonucleotide sequence. The duplexes apparently interact only through their diffuse ionic atmospheres. The results obtained can thus be considered as intermediate between liquid crystals, fibers, and standard crystal structures. They provide new information on medium range DNA-DNA interactions.     

Individual-based modelling of bacterial cultures to study the microscopic causes of the lag phase

The lag phase has been widely studied for years in an effort to contribute to the improvement of food safety. Many analytical models have been built and tested by several authors. The use of Individual-based Modelling (IbM) allows us to probe deeper into the behaviour of individual cells; it is a bridge between theories and experiments when needed. INDividual DIScrete SIMulation (INDISIM) has been developed and coded by our group as an IbM simulator and used to study bacterial growth, including the microscopic causes of the lag phase. First of all, the evolution of cellular masses, specifically the mean mass and biomass distribution, is shown to be a determining factor in the beginning of the exponential phase. Secondly, whenever there is a need for an enzyme synthesis, its rate has a direct effect on the lag duration. The variability of the lag phase with different factors is also studied. The known decrease of the lag phase with an increase in the temperature is also observed in the simulations. An initial study of the relationship between individual and collective lag phases is presented, as a complement to the studies already published. One important result is the variability of the individual lag times and generation times. It has also been found that the mean of the individual lags is greater than the population lag. This is the first in a series of studies of the lag phase that we are carrying out. Therefore, the present work addresses a generic system by making a simple set of assumptions.     

Microsatellite loci for Paspalum atratum (Poaceae) and cross-amplification in other species

? Premise of the study: Paspalum atratum is a perennial, cespitose, tropical grass native to Central and South America. This species belongs to a polyploid complex (Plicatula group) little known at the genetic level. The characterized microsatellite markers provide new informative tools for further studies of the hybridization, mating systems, and structure of the population. ? Methods and Results: Using the microsatellite-enriched library method, we isolated and characterized 19 microsatellite markers from P. atratum. Eleven of them were polymorphic, showing a variable degree of variation, while eight were monomorphic in the samples analyzed. Additionally, the transferability of these microsatellite markers was tested in other species. ? Conclusions: These results suggest that the characterized markers have enough discriminatory potential to be used in genetic characterizations of Paspalum taxa, which are based on an understanding of their mating systems and genetic structure, as well as in understanding the evolutionary processes involved in the evolution of groups of Paspalum.     

Polymorphisms in LEP and NPY genes modify the response to soluble fibre Plantago ovata husk intake on cardiovascular risk biomarkers

The satiating effect of fibre consumption has been related to gut hormones, such as peptide YY and leptin. These peptides may also influence cardiovascular (CVD) risk biomarkers. Nevertheless, there is wide interindividual variation in metabolic responses to fibre consumption. The objective was to investigate differences in the effects of soluble fibre, in the form of Plantago ovata husk (Po-husk) treatment, on CVD risk biomarkers according to selected polymorphisms in genes related to satiety. The study was a multi-centred, double-blind, placebo-controlled, parallel and randomised trial in mild–moderate hypercholesterolaemic patients (age range: 43–67 years). Eight polymorphisms in three genes related to satiety (LEP, NPY and PYY) were identified in 178 participants; 88 patients in the placebo (microcrystalline cellulose 14 g/day) group and 90 in the Po-husk (14 g/day) group, which had added to a low-saturated-fat diet for 8 weeks. The CVD biomarkers measured included the following: lipid profile, blood pressure (BP), glucose, insulin, hs-CRP, oxidised LDL and IL-6. Relative to the placebo, Po-husk consumption lowered the plasma total cholesterol concentration by 3.3 % according to rs7799039 polymorphism in the LEP gene (p < 0.05). Furthermore, the Po-husk reduced systolic BP (mean [95 % CI]) by −8 mmHg (−14.16; −1.90) and hs-CRP by 24.9 % in subjects with the AA genotype of the rs16147 polymorphism in the NPY gene (32 % of our total population; p < 0.05), which remained significant after Bonferroni correction. In conclusion, polymorphisms in the LEP and NPY genes potentiate the response to Po-husk, particularly the effects on systolic BP and the hs-CRP plasma concentration.     

Dobutamine in Paediatric Population: A Systematic Review in Juvenile Animal Models

Objective

Although dobutamine is widely used in neonatal clinical practice, the evidence for its use in this specific population is not clear. We conducted a systematic review of the use of dobutamine in juvenile animals to determine whether the evidence from juvenile animal experiments with dobutamine supported the design of clinical trials in neonatal/paediatric population.

Methods

Studies were identified by searching MEDLINE (1946–2012) and EMBASE (1974–2012). Articles retrieved were independently reviewed by three authors and only those concerning efficacy and safety of the drug in juvenile animals were included. Only original articles published in English and Spanish were included.

Results

Following our literature search, 265 articles were retrieved and 24 studies were included in the review: 17 focused on neonatal models and 7 on young animal models. Although the aims and design of these studies, as well as the doses and ages analysed, were quite heterogeneous, the majority of authors agree that dobutamine infusion improves cardiac output in a dose dependent manner. Moreover, the cardiovascular effects of dobutamine are influenced by postnatal age, as well as by the dose used and the duration of the therapy. There is inadequate information about the effects of dobutamine on cerebral perfusion to draw conclusions.

Conclusion

There is enough preclinical evidence to ensure that dobutamine improves cardiac output, however to better understand its effects in peripheral organs, such as the brain, more specific and well designed studies are required to provide additional data to support the design of clinical trials in a paediatric population.     

Cocoa, hazelnuts, sterols and soluble fiber cream reduces lipids and inflammation biomarkers in hypertensive patients: a randomized controlled trial

Background

Cocoa, mixed with other food ingredients, intake can have beneficial effects on cardiovascular disease (CVD) biomarkers. We compared the effects of 4 cocoa cream products on some of these biomarkers.

Methods and Findings

In this multi-centered, randomized, controlled, double-blind, parallel trial, volunteers (n = 113; age range: 43–65 years) who were pre-hypertensive, stage-1 hypertensive and hypercholesterolemic received one of 4 cocoa cream products (13 g/unit; 1 g cocoa/unit, 6 units/d; 465 Kcal/d) added to a low saturated fat diet for 4 weeks. The groups were: A) (n = 28), cocoa cream considered as control; B) (n = 28), cocoa+hazelnut cream (30 g/d hazelnuts); C) (n = 30), cocoa+hazelnuts+phytosterols (2 g/d); and D) (n = 27), cocoa+hazelnuts+phytosterols+soluble fiber (20 g/d) the patented “LMN product”. Primary outcome measures were BP, LDL-c, apolipoprotein B-100 (Apo B), ApoB/ApoA ratio, oxidized LDL (oxLDL) and high-sensitive C-reactive protein (hsCRP) determined at baseline and post-cocoa cream product intake. Statistical analysis used was ANCOVA or mixed models (in case of repeated measurements), with baseline observation included as a covariate. After 4 weeks, compared to product A, product C reduced LDL-c by 11.2%, Apo B by 8.1% and ApoB/ApoA ratio by 7.8% (P = 0.01). LMN decreased LDL-c by 9.2%, Apo B-100 by 8.5%, ApoB/ApoA ratio by 10.5%, hsCRP by 33.4% and oxLDL by 5.9% (P = 0.01). Surprisingly, even “control” product A reduced systolic BP (−7.89 mmHg; 95%CI: −11.45 to −4.3) and diastolic BP (−5.54 mmHg; 95%CI: −7.79 to −3.29). The BP reductions were similar with the other 3 products. Limitations of the study are that the trial period was relatively short and that a better “BP control” product would have been preferable.

Conclusion

The creams (particularly the LMN) have anti-inflammatory and antioxidant effects in addition to lowering LDL-c, Apo B and ApoB/ApoA ratio. Thus, the soluble fiber effects amplified with sterols (as contained in the cocoa creams) provide new dietary therapeutic perspectives.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00511420","term_id":"NCT00511420"}}NCT00511420     

Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine- protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu(++)-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.     

The Role of Glutathione in Protection against DNA Damage Induced by Rifamycin SV And Copper(II) Ions

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH·) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively.

Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.