A synthetic biochemistry platform for cell free production of monoterpenes from glucose.

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals

Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme.

Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes

Bacteriorhodopsin folds through a poorly organized transition state.

The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations

An Adaptation To Life In Acid Through A Novel Mevalonate Pathway.

Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments

Risk factors for chest infection in acute stroke: a prospective cohort study

<p><b>Background and Purpose:</b> Pneumonia is a major cause of morbidity and mortality after stroke. We aimed to determine key characteristics that would allow prediction of those patients who are at highest risk for poststroke pneumonia.</p> <p><b>Methods:</b> We studied a series of consecutive patients with acute stroke who were admitted to hospital. Detailed evaluation included the modified National Institutes of Health Stroke Scale; the Abbreviated Mental Test; and measures of swallow, respiratory, and oral health status. Pneumonia was diagnosed by set criteria. Patients were followed up at 3 months after stroke.</p> <p><b>Results:</b> We studied 412 patients, 391 (94.9%) with ischemic stroke and 21 (5.1%) with hemorrhagic stroke; 78 (18.9%) met the study criteria for pneumonia. Subjects who developed pneumonia were older (mean±SD age, 75.9±11.4 vs 64.9±13.9 years), had higher modified National Institutes of Health Stroke Scale scores, a history of chronic obstructive pulmonary disease, lower Abbreviated Mental Test scores, and a higher oral cavity score, and a greater proportion tested positive for bacterial cultures from oral swabs. In binary logistic-regression analysis, independent predictors (P<0.05) of pneumonia were age >65 years, dysarthria or no speech due to aphasia, a modified Rankin Scale score ≥4, an Abbreviated Mental Test score <8, and failure on the water swallow test. The presence of 2 or more of these risk factors carried 90.9% sensitivity and 75.6% specificity for the development of pneumonia.</p> <p><b>Conclusions:</b> Pneumonia after stroke is associated with older age, dysarthria/no speech due to aphasia, severity of poststroke disability, cognitive impairment, and an abnormal water swallow test result. Simple assessment of these variables could be used to identify patients at high risk of developing pneumonia after stroke.</p&gt

Author Correction: A cell-free platform for the prenylation of natural products and application to cannabinoid production.

In the original version of this Article, the genotype of the M30 mutant presented in Fig. 3b was given incorrectly as Y288V/A232S, and the M31 mutant was given incorrectly as M1/A232S. The correct genotype of the M30 mutant is Y288A/A232S and for M31 it is Y288V/A232S. In addition, to keep consistency in genotype formatting, the genotype of the M27 mutant should be Y288V/G286S. The errors have been corrected in both the PDF and HTML versions of the Article

Conservation and biology of the rediscovered nationally endangered Canterbury knobbled weevil, Hadramphus tuberculatus

Three areas near Burkes Pass Scenic Reserve were surveyed for the presence of Hadramphus tuberculatus, a recently rediscovered endangered weevil. The reserve itself was resurveyed to expand on a 2005/2006 survey. Non-lethal pitfall traps and mark and recapture methods were used. Six H. tuberculatus were caught in pitfall traps over 800 trap nights. Day and night searching of Aciphylla aurea was conducted. Four specimens were observed on Aciphylla flowers between 9 am and 1.30 pm within the reserve. No specimens were found outside of the reserve by either method. Other possible locations where H. tuberculatus may be found were identified and some visited. At most locations Aciphylla had already finished flowering, no H. tuberculatus were found. Presence of H. tuberculatus at other sites would be best determined by searching of Aciphylla flowers during the morning from late October onwards

Can we quantify harm in general practice records? An assessment of precision and power using computer simulation

<b>Background</b> Estimating harm rates for specific patient populations and detecting significant changes in them over time are essential if patient safety in general practice is to be improved. Clinical record review (CRR) is arguably the most suitable method for these purposes, but the optimal values and combinations of its parameters (such as numbers of records and practices) remain unknown. Our aims were to: 1. Determine and quantify CRR parameters; 2. Assess the precision and power of feasible CRR scenarios; and 3. Quantify the minimum requirements for adequate precision and acceptable power.<p></p> <b>Method</b> We explored precision and power of CRR scenarios using Monte Carlo simulation. A range of parameter values were combined in 864 different CRR scenarios, 1000 random data sets were generated for each, and harm rates were estimated and tested for change over time by fitting a generalised linear model with a Poisson response.<p></p> <b>Results</b> CRR scenarios with ≥100 detected harm incidents had harm rate estimates with acceptable precision. Harm reductions of 20% or ≥50% were detected with adequate power by those CRR scenarios with at least 100 and 500 harm incidents respectively. The number of detected harm incidents was dependent on the baseline harm rate multiplied by: the period of time reviewed in each record; number of records reviewed per practice; number of practices who reviewed records; and the number of times each record was reviewed.<p></p> <b>Conclusion</b> We developed a simple formula to calculate the minimum values of CRR parameters required to achieve adequate precision and acceptable power when monitoring harm rates. Our findings have practical implications for health care decision-makers, leaders and researchers aiming to measure and reduce harm at regional or national level