The pPSU Plasmids for Generating DNA Molecular Weight Markers.

Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications

Cutinase from Amycolatopsis mediterannei: marked activation and stabilisation in Deep Eutectic Solvents

Amycolatopsis mediterranei cutinase (AmCut) has potential biocatalytic applications in plastics degradation and ester synthesis. Deep Eutectic Solvents (DES) are next generation biodegradable solvents for biocatalysis. However, the behaviour of cutinase enzymes in DES is little studied. Herein, we examine the effect of selected DES, and their components, on AmCut activity and stability. Low amounts (10% v/v) of DES (choline chloride:glycerol; 1:1 mole ratio) caused striking activation of AmCut (over 2-fold). Further examination showed that the choline chloride component of DES caused the observed activation. This is the first report of activation of a cutinase by a small molecule. At higher concentrations (50% v/v), DES composed of a choline chloride with glycerol as hydrogen bond donor dramatically increased the thermostability of AmCut - the enzyme lost no activity after incubation at 50oC for 2 hours. The biotechnological utility and physiological relevance of choline chloride activation and stabilisation is discussed

Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis. The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway. E. coli is a useful host for recombinant enzymes used in biocatalysis due to the ease of genetic manipulation in this organism, which allows tuning of enzymes for specific applications, by mutagenesis. When a truncated AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in the periplasmic space and in the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (A. mediterranei) was being recognised by the Gram-negative (E. coli) Sec transmembrane transport system. AmCut was transported into E. coli’s periplasmic space from where it was released into the culture medium. Although the periplasmic targeting was surprising, it is not unprecedented due to the conservation of the Sec pathway across species. It was more surprising that AmCut was secreted from the periplasmic space into the culture medium. This suggests that extracellular AmCut translocation across the E. coli outer membrane may involve non-classical secretion pathways. This tuneable recombinant E. coli expressing extracellular AmCut may be useful for degradation of polyester substrates in the environment; this and other applications are discussed

An Extracellular Lipase from Amycolatopsis Mediterannei is a Cutinase with Plastic Degrading Activity

An extracellular lipase from Amycolatopsis mediteranei (AML) with potential applications in process biotechnology was recently cloned and examined in this laboratory. In the present study, the 3D structure of AML was elucidated by comparative modelling. AML lacked the ‘lid’ structure observed in most true lipases and shared similarities with plastic degrading enzymes. Modelling and substrate specificity studies showed that AML was a cutinase with a relatively exposed active site and specificity for medium chain fatty acyl moieties. AML rapidly hydrolysed the aliphatic plastics poly(ε-caprolactone) and poly(1,4-butylene succinate) extended with 1,6-diisocyanatohexane under mild conditions. These plastics are known to be slow to degrade in landfill. Poly(L-lactic acid) was not hydrolysed by AML, nor was the aromatic plastic Polyethylene Terephthalate (PET). The specificity of AML is partly explained by active site topology and analysis reveals that minor changes in the active site region can have large effects on substrate preference. These findings show that extracellular Amycolatopsis enzymes are capable of degrading a wider range of plastics than is generally recognised. The potential for application of AML in the bioremediation of plastics is discussed

Parkinson\u27s disease and the gastrointestinal microbiome

Recently, there has been a surge in awareness of the gastrointestinal microbiome (GM) and its role in health and disease. Of particular note is an association between the GM and Parkinson’s disease (PD) and the realisation that the GM can act via a complex bidirectional communication between the gut and the brain. Compelling evidence suggests that a shift in GM composition may play an important role in the pathogenesis of PD by facilitating the characteristic ascending neurodegenerative spread of α-synuclein aggregates from the enteric nervous system to the brain. Here, we review evidence linking GM changes with PD, highlighting mechanisms supportive of pathological α-synuclein spread and intestinal inflammation in PD. We summarise existing patterns and correlations seen in clinical studies of the GM in PD, together with the impacts of non-motor symptoms, medications, lifestyle, diet and ageing on the GM. Roles of GM modulating therapies including probiotics and faecal microbiota transplantation are discussed. Encouragingly, alterations in the GM have repeatedly been observed in PD, supporting a biological link and highlighting it as a potential therapeutic target

Two distinct halo populations in the solar neighborhood. IV. Lithium abundances

We investigate if there is a difference in the lithium abundances of stars belonging to two halo populations of F and G main-sequence stars previously found to differ in [alpha/Fe] for the metallicity range -1.4 < [Fe/H] < -0.7. Li abundances are derived from the LiI 6707.8 A line measured in high-resolution spectra using MARCS model atmospheres. Furthermore, masses of the stars are determined from the logTeff - logg diagram by interpolating between Yonsei-Yale evolutionary tracks. There is no significant systematic difference in the lithium abundances of high- and low-alpha halo stars. For the large majority of stars with masses 0.7 < M/M_sun < 0.9 and heavy-element mass fractions 0.001 < Z < 0.006, the Li abundance is well fitted by a relation A(Li) = a0 + a1 M + a2 Z + a3 M Z, where a0, a1, a2, and a3 are constants. Extrapolating this relation to Z = 0 leads to a Li abundance close to the primordial value predicted from standard Big Bang nucleosynthesis calculations and the WMAP baryon density. The relation, however, does not apply to stars with [Fe/H] < -1.5. We suggest that metal-rich halo stars were formed with a Li abundance close to the primordial value, and that lithium in their atmospheres has been depleted in time with an approximately linear dependence on stellar mass and Z. The lack of a systematic difference in the Li abundances of high- and low-alpha stars indicates that an environmental effect is not important for the destruction of lithium.Comment: 10 pages, 7 figures, accepted for publication in Astronomy and Astrophysic

1999 Media Guide

1999 Men\u27s Track and Field Media Guide, George Fox College

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUS(R521G), harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS

Biological Conversion of Sugars to Hydrocarbons Technology Pathway

In support of the Bioenergy Technologies Office, the National Renewable Energy Laboratory (NREL) and the Pacific Northwest National Laboratory (PNNL) are undertaking studies of biomass conversion technologies to identify barriers and target research toward reducing conversion costs. Process designs and preliminary economic estimates for each of these pathway cases were developed using rigorous modeling tools (Aspen Plus and Chemcad). These analyses incorporated the best information available at the time of development, including data from recent pilot and bench-scale demonstrations, collaborative industrial and academic partners, and published literature and patents. This technology pathway case investigates the biological conversion of biomass derived sugars to hydrocarbon biofuels, utilizing data from recent literature references and information consistent with recent pilot scale demonstrations at NREL. Technical barriers and key research needs have been identified that should be pursued for the pathway to become competitive with petroleum-derived gasoline, diesel and jet range hydrocarbon blendstocks