Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing.

BACKGROUND: During the kraft process to obtain cellulosic pulp from wood, most of the lignin is removed by high-temperature alkaline cooking, released in the black liquors and usually incinerated for energy. However, kraft lignins are a valuable source of phenolic compounds that can be valorized in new bio-based products. The aim of this work is to develop laccases capable of working under the extreme conditions of high temperature and pH, typical of the industrial conversion of wood into kraft pulp and fibreboard, in order to provide extremophilic biocatalysts for depolymerising kraft lignin, and enzyme-assisted technologies for kraft pulp and fibreboard production. RESULTS: Through systematic enzyme engineering, combining enzyme-directed evolution and rational design, we changed the optimal pH of the laccase for oxidation of lignin phenols from acidic to basic, enhanced the catalytic activity at alkaline pH and increased the thermal tolerance of the enzyme by accumulating up to eight mutations in the protein sequence. The extremophilic laccase variants show maximum activity at 70 °C and oxidize kraft lignin at pH 10. Their integration into industrial-type processes saves energy and chemicals. As a pre-bleaching stage, the enzymes promote kraft pulp bleachability and significantly reduce the need for chlorine dioxide compared to the industrial sequence. Their application in wood chips during fibreboard production, facilitates the defibering stage, with less energy required. CONCLUSIONS: A set of new alkaliphilic and thermophilic fungal laccases has been developed to operate under the extreme conditions of high temperature and pH typical of industrial wood conversion processes. For the first time basidiomycete laccases of high-redox potential show activity on lignin-derived phenols and polymeric lignin at pH 10. Considering the extreme conditions of current industrial processes for kraft pulp and fibreboard production, the new tailor-made laccases constitute a step forward towards turning kraft pulp mills into biorefineries. Their use as biocatalysts in the wood conversion sector is expected to support the development of more environmentally sound and efficient processes, and more sustainable products.

Use of a Novel Extremophilic Xylanase for an Environmentally Friendly Industrial Bleaching of Kraft Pulps.

Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology.

Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC). RESULTS: The fungal LPMO from AA9 family (PaLPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with PaLPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC. CONCLUSIONS: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.

Genotoxic and inflammatory effects of nanofibrillated cellulose in murine lungs.

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300-1000nm, thickness 10-25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1ß and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.

First-trimester combined screening for trisomy 21 in women with renal disease.

OBJECTIVE: To evaluate the results of first-trimester combined screening for Down syndrome in women with chronic renal disease. METHOD: Fifty-five pregnant women with renal disease were compared with 110 patients matched for maternal age, maternal weight, smoking status, and gestational age. Maternal renal function was assayed at the time of the combined screening, and renal insufficiency was defined by serum creatinine >90 µmol/L and renal clearance <80 mL/min. We defined three groups: kidney disease and normal renal function (group 1), kidney disease and renal insufficiency (group 2), and a control group (group 3). The values of nuchal translucency, pregnancy-associated plasma protein A, human ß-chorionic gonadotrophin (hCGß), and false-positive rates for Down syndrome screening were compared. RESULTS: There were 39 (71%) and 16 (29%) cases in groups 1 and 2, respectively. Nuchal translucency and multiple of the median (MoM) pregnancy-associated plasma protein A were similar in the three groups. However, MoM hCGß levels were higher in group 2 than in groups 1 and 3 (5.37 vs 1.1 vs 0.98 MoM, p = 0.0001). The resulting screen-positive rate was also higher in group 2 than in groups 1 and 3 (43.7% vs 10.2% vs 5.5%, p = 0.0001). CONCLUSION: Trisomy 21 first-trimester screening using hCGß is not suitable in the case of maternal renal failure. © 2014 John Wiley & Sons, Ltd.

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization.

Design, synthesis and biological evaluation of trimethine cyanine dyes as fluorescent probes for the detection of tau fibrils in Alzheimer's disease brain and olfactory epithelium.

Shedding light on grey matter: Fluorescent trimethine cyanines were evaluated as imaging probes for neurofibrillary tangles in post-mortem brain sections of Alzheimer's disease patients. These probes bind to neurofibrillary tangles with high contrast and selectivity over amyloid ß plaques.

2-Styrylindolium based fluorescent probes visualize neurofibrillary tangles in Alzheimer's disease.

We evaluated 2-styrylindolium derivatives (6-11) as novel and selective probes for neurofibrillary tangles (NFTs) on brain sections of AD patients. The staining experiments indicated that these compounds may bind selectively to NFTs in the presence of ß-amyloid (Aß) plaques. Cell free binding assays confirmed that 2-[2-[4-(1-pyrrolidinyl)phenyl]ethenyl]-1,3,3-trimethyl-3H-indolium iodide (9) and 2-[2-[4-(diethylamino)phenyl]ethenyl]-1-butyl-3,3-dimethyl-3H-indolium iodide (11) display excellent affinities to Tau-aggregates (IC(50) values of 5.1 and 1.4 nM, respectively) in the displacement of Thiazin Red R. These probes have good solubility in distilled water and low or no cytotoxicity in zebrafish embryo and liver hepatocellular carcinoma cell assays.

Laccase/HBT and laccase-CBM/HBT treatment of softwood kraft pulp: impact on pulp bleachability and physical properties.

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments.

Risk and pharmacoeconomic analyses of the injectable medication process in the paediatric and neonatal intensive care units.

OBJECTIVE: To analyse safety risks in injectable medications. To assess the potential impact and pharmacoeconomic aspects of safety tools. DESIGN: The injectable drug process was prospectively assessed using a failure modes, effects and criticality analysis. Criticality indexes were estimated based on their likelihood of occurrence, detection probability and potential severity. The impact of 10 safety tools on the criticality index was calculated and extrapolated to all drugs injected daily. Yearly costs for a reduction in criticality by 1 point (=1 quali) per day were estimated. SETTING: Paediatric and neonatal intensive care units in a University Hospital. PARTICIPANTS: Two paediatric nurses, a neonatologist, three hospital pharmacists. INTERVENTIONS: Qualitative and quantitative risk assessment. MAIN OUTCOME MEASURES: Failure modes, criticality indexes, cost-efficacy ratios. RESULTS: Thirty-one failure modes identified, with the mean of their entire criticality indexes totalling 4540. The most critical failure mode was microbial contamination. The following gains were predicted: 1292 quali (46 500 per day by extrapolation) from ready-to-use syringes, 1201 (72 060) by employing a clinical pharmacist, 996 (59 780) from double check by nurses and 984 (59 040) with computerized physician order entry. The best cost-efficacy ratios were obtained for a clinical pharmacist (1 quali = 0.54 euros), double check (1 quali = 0.71 euros) and ready-to-use syringes (1 quali = 0.72 euros). Computerized physician order entry showed the worst cost-efficacy ratio due to a very high investment costs (1 quali = 22.47 euros). CONCLUSION: Based on our risk and pharmacoeconomic analyses, clinical pharmacy and ready-to-use syringes appear as the most promising safety tools.