Expression optimisation of recombinant α-Larabinofuranosidase from Aspergillus niger ATCC 120120 in Pichia pastoris and its biochemical characterisation

A gene encoding α-L-arabinofuranosidase (AnabfA) from Aspergillus niger ATCC 120120 was successfully cloned and expressed in Pichia pastoris under the control of the AOX1 promoter. The effect of cultural conditions on recombinant AnabfA production was studied and the enzyme was expressed as a soluble protein. Recombinant AnabfA was expressed as an active enzyme at 28°C when cultured in BMMY medium (pH 6.0) and induced with 2% methanol every 24 h. Maximum activity was observed 5 days after induction. The purified recombinant AnabfA before and after treatment with PNGase F migrated by SDS-PAGE had relative molecular masses of about 83 and 66 kDa, respectively, suggesting that the AnabfA contains N-linked oligosaccharides. Characterisation of the purified recombinant AnabfA showed an optimum temperature and pH of 50°C and 4, respectively. The enzyme was stable at a pH of 3 to 6 and retained more than 80% of its activity after pre-incubation at 40°C for 30 min. The recombinant AnabfA activity was stimulated by K+, Mn2+, Na2+ and triton X-100 and was strongly inhibited by Cu2+ and Fe2+ and the enzyme activity was relatively unaffected by Ca2+, CO2+, Mg2+ and EDTA. The Km and Vmax of the purified recombinant AnabfA activity towards ρNPA were 0.93 mM and 17.86 μmol/ml/min, respectively.Key words: Aspergillus niger, α-L-arabinofuranosidase, expression, Pichia pastoris, characterisation

Antimicrobial Resistance Profiles Diversity in Salmonella from Humans Cattle, 2004-2011

Analysis of long-term anti-microbial resistance (AMR) data is useful to understsource transmission dynamics of AMR. We analysed 5124 human clinical isolates from Washington State Department of Health, 391 cattle clinical isolates from the Washington Animal Disease Diagnostic Laboratory 1864 non-clinical isolates from foodborne disease research on dairies in the Pacific Northwest. Isolates were assigned profiles based on phenotypic resistance to 11 anti-microbials belonging to eight classes. Salmonella Typhimurium (ST), Salmonella Newport (SN) Salmonella Montevideo (SM) were the most common serovars in both humans cattle. Multinomial logistic regression showed ST SN from cattle had greater probability of resistance to multiple classes of anti-microbials than ST SN from humans (P < 0.0001). While these findings could be consistent with the belief that cattle are a source of resistant ST SN for people, occurrence of profiles unique to cattle not observed in temporally related human isolates indicates these profiles are circulating in cattle only. We used various measures to assess AMR diversity, conditional on the weighting of rare versus abundant profiles. AMR profile richness was greater in the common serovars from humans, although both source data sets were dominated by relatively few profiles. The greater profile richness in human Salmonella may be due to greater diversity of sources entering the human population compared to cattle or due to continuous evolution in the human environment. Also, AMR diversity was greater in clinical compared to non-clinical cattle Salmonella, this could be due to anti-microbial selection pressure in diseased cattle that received treatment. The use of bootstrapping techniques showed that although there were shared profiles between humans cattle, the expected observed number of profiles was different, suggesting Salmonella associated resistance from humans cattle may not be wholly derived from a common population

Update on emergency contraception

pre-printEmergency contraception (EC) is any method used after sexual intercourse to prevent pregnancy. This article provides an overview of the history of EC methods and describes the current availability of oral and intrauterine EC. Oral forms include the Yuzpe regimen (combining ethinyl estradiol and levonorgestrel), levonorgestrel-only pills, and ulipristal acetate, which is a new emergency contraceptive drug recently approved by the US Food and Drug Administration. The copper T-380A intrauterine device can also be used for EC. Information about dosing, timing, access, and other considerations in the provision of EC is covered. Clinicians should be aware of all available options in order to counsel women in need of EC appropriately

Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris>/i>

Cellulases are industrially important hydrolytic enzymes applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, an endoglucanase from Aspergillus niger ATCC 10574, EglA, was expressed in the methylotrophic yeast Pichia pastoris and the properties of the recombinant protein were characterized. The full length cDNA of eglA has been cloned into a pPICZαC expression vector and expressed extracellularly as a ~30 kDa recombinant protein in P. pastoris X-33. Pure EglA displayed optimum activity at 50°C and was stable between 30 and 55°C. The pH stability of this enzyme was shown to be in the range of pH 2.0 to 7.0 and optimum at pH 4.0. EglA showed the highest affinity toward β-glucan followed by carboxymethyl cellulose (CMC) with a specific activity of 63.83 and 9.47 U/mg, respectively. Very low or no detectable hydrolysis of cellobiose, laminarin, filter paper and avicel were observed. Metal ions such as Mn2+, Co2+, Zn2+, Mg2+, Ba2+, Fe2+, Ca2+ and K+ showed significant augmentation of endoglucanase activity, with manganese ions causing the highest increase in activity to about 2.7 fold when compared with the control assay, whereas Pd2+, Cu2+, SDS and EDTA showed inhibition of EglA activity.Key words: Cellulase, endoglucanase, recombinant, Aspergillus niger, Pichia pastoris

Nishimori point in random-bond Ising and Potts models in 2D

We study the universality class of the fixed points of the 2D random bond q-state Potts model by means of numerical transfer matrix methods. In particular, we determine the critical exponents associated with the fixed point on the Nishimori line. Precise measurements show that the universality class of this fixed point is inconsistent with percolation on Potts clusters for q=2, corresponding to the Ising model, and q=3Comment: 11 pages, 3 figures. Contribution to the proceedings of the NATO Advanced Research Workshop on Statistical Field Theories, Como 18-23 June 200

Magnetic resonance imaging quantification of fasted state colonic liquid pockets in healthy humans

The rate and extent of drug dissolution and absorption from solid oral dosage forms is highly dependent on the volume of liquid in the gastrointestinal tract (GIT). However, little is known about the time course of GIT liquid volumes after drinking a glass of water (8 oz), particularly in the colon, which is a targeted site for both locally and systemically acting drug products. Previous magnetic resonance imaging (MRI) studies offered novel insights on GIT liquid distribution in fasted humans in the stomach and small intestine, and showed that freely mobile liquid in the intestine collects in fairly distinct regions or “pockets”. Based on this previous pilot data, we hypothesized that (1) it is possible to quantify the time course of the volume and number of liquid pockets in the undisturbed colon of fasted healthy humans following ingestion of 240 mL, using noninvasive MRI methods; (2) the amount of freely mobile water in the fasted human colon is of the order of only a few milliliters. Twelve healthy volunteers fasted overnight and underwent fasted abdominal MRI scans before drinking 240 mL (∼8 fluid ounces) of water. After ingesting the water they were scanned at frequent intervals for 2 h. The images were processed to quantify freely mobile water in the total and regional colon: ascending, transverse, and descending. The fasted colon contained (mean ± SEM) 11 ± 5 pockets of resting liquid with a total volume of 2 ± 1 mL (average). The colonic fluid peaked at 7 ± 4 mL 30 min after the water drink. This peak fluid was distributed in 17 ± 7 separate liquid pockets in the colon. The regional analysis showed that pockets of free fluid were found primarily in the ascending colon. The interindividual variability was very high; the subjects showed a range of number of colonic fluid pockets from 0 to 89 and total colonic freely mobile fluid volume from 0 to 49 mL. This is the first study measuring the time course of the number, regional location, and volume of pockets of freely mobile liquid in the undisturbed colon of fasted humans after ingestion of a glass of water. Novel insights into the colonic fluid environment will be particularly relevant to improve our understanding and design of the in vivo performance of controlled release formulations targeted to the colon. The in vivo quantitative information presented here can be input into physiologically based mechanistic models of dissolution and absorption, and can be used in the design and set up of novel in vitro performance tools predictive of the in vivo environment

A single low-energy, iron-poor supernova as the source of metals in the star SMSS J 031300.36-670839.3

The element abundance ratios of four low-mass stars with extremely low metallicities indicate that the gas out of which the stars formed was enriched in each case by at most a few, and potentially only one low-energy, supernova. Such supernovae yield large quantities of light elements such as carbon but very little iron. The dominance of low-energy supernovae is surprising, because it has been expected that the first stars were extremely massive, and that they disintegrated in pair-instability explosions that would rapidly enrich galaxies in iron. What has remained unclear is the yield of iron from the first supernovae, because hitherto no star is unambiguously interpreted as encapsulating the yield of a single supernova. Here we report the optical spectrum of SMSS J031300.36- 670839.3, which shows no evidence of iron (with an upper limit of 10^-7.1 times solar abundance). Based on a comparison of its abundance pattern with those of models, we conclude that the star was seeded with material from a single supernova with an original mass of ~60 Mo (and that the supernova left behind a black hole). Taken together with the previously mentioned low-metallicity stars, we conclude that low-energy supernovae were common in the early Universe, and that such supernovae yield light element enrichment with insignificant iron. Reduced stellar feedback both chemically and mechanically from low-energy supernovae would have enabled first-generation stars to form over an extended period. We speculate that such stars may perhaps have had an important role in the epoch of cosmic reionization and the chemical evolution of early galaxies.Comment: 28 pages, 6 figures, Natur

The need for education on health related-quality of life

<p>Abstract</p> <p>Background</p> <p>Health-related quality of life is increasingly recognised as an important outcome measure that complements existing measures of clinical effectiveness. The education available on this subject for different healthcare professionals is varied. This article describes the design, implementation and evaluation of a Special Study Module on Health-Related Quality of Life for undergraduate medical students at the University of Birmingham.</p> <p>Methods</p> <p>The course involves 10 hours of "guided discovery learning" covering core concepts of Health-Related Quality of Life assessment including methodological considerations, use in clinical trials, routine practice and in health policy followed by self-directed learning. The taught components aim to provide students with the skills and knowledge to enable them to explore and evaluate the use of quality of life assessments in a particular patient group, or setting, through self-directed learning supported by tutorials.</p> <p>Results</p> <p>The use of case studies, recent publications and research, and discussion with a research oncology nurse in task-based learning appeared to provide students with a stimulating environment in which to develop their ideas and was reflected in the diverse range of subjects chosen by students for self-directed study and the positive feedback on the module. Course evaluation and student assessment suggests that quality of life education appears to integrate well within the medical curriculum and allows students to develop and utilise skills of time-management and independent, self-directed learning that can be applied in any context.</p> <p>Conclusion</p> <p>We suggest that education and training initiatives in quality of life may improve the quality of studies, and help bridge the gap between research and clinical practice. Resources for curriculum development on health-related quality of life have been developed by the International Society for Quality of Life Research and may prove a useful tool to educators interested in this area.</p

Two Stellar Components in the Halo of the Milky Way

The halo of the Milky Way provides unique elemental abundance and kinematic information on the first objects to form in the Universe, which can be used to tightly constrain models of galaxy formation and evolution. Although the halo was once considered a single component, evidence for its dichotomy has slowly emerged in recent years from inspection of small samples of halo objects. Here we show that the halo is indeed clearly divisible into two broadly overlapping structural components -- an inner and an outer halo -- that exhibit different spatial density profiles, stellar orbits and stellar metallicities (abundances of elements heavier than helium). The inner halo has a modest net prograde rotation, whereas the outer halo exhibits a net retrograde rotation and a peak metallicity one-third that of the inner halo. These properties indicate that the individual halo components probably formed in fundamentally different ways, through successive dissipational (inner) and dissipationless (outer) mergers and tidal disruption of proto-Galactic clumps.Comment: Two stand-alone files in manuscript, concatenated together. The first is for the main paper, the second for supplementary information. The version is consistent with the version published in Natur