High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Performance of the LHCb RICH detector at the LHC.

The LHCb experiment has been taking data at the Large Hadron Collider (LHC) at CERN since the end of 2009. One of its key detector components is the Ring-Imaging Cherenkov (RICH) system. This provides charged particle identification over a wide momentum range, from 2-100 GeV/c. The operation and control, software, and online monitoring of the RICH system are described. The particle identification performance is presented, as measured using data from the LHC. Excellent separation of hadronic particle types (π, K, p) is achieved.

Combined positron emission tomography/computed tomography in sunitinib therapy assessment of patients with metastatic renal cell carcinoma.

AIM: To assess the clinical benefit of combined functional imaging with [(18)F]2-fluoro-2-deoxy-d-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) in patients with metastatic renal cell carcinoma (mRCC) treated with the tyrosine kinase inhibitor sunitinib. MATERIALS AND METHODS: Fourteen patients with mRCC were prospectively enrolled in this study. All patients underwent PET/CT before receiving at least two cycles of sunitinib treatment. Three months after the onset of sunitinib treatment, a second PET/CT was carried out. The metabolic response evaluated from the PET (standard uptake value; SUV) was compared with the CT component of the PET/CT. The Response Evaluation Criteria in Solid Tumours criteria were used to assess the CT response and modified European Organization for Research and Treatment of Cancer criteria were used to assess the PET response. RESULTS: Three main results were obtained: (1) Patients with relatively low 18F-FDG uptake before treatment (SUV<5) had a longer progression-free survival than those with a relatively high 18F-FDG uptake (P=0.006). (2) Patients with a partial metabolic response or stable metabolic disease after two courses of sunitinib had improved prognosis as compared with those with progressive metabolic disease (P=0.031). (3) There was a clear discrepancy between PET and CT as a tool for the evaluation of treatment response after two courses of sunitinib. PET indicated progressive disease in three patients, a partial response in six patients and stable disease in four patients. In contrast, CT concluded with progression in only one patient and stable disease in all other patients. CONCLUSION: In patients with mRCC, a high baseline 18F-FDG uptake indicates aggressive disease, and the degree of reduction in 18F-FDG uptake after sunitinib treatment adds valuable prognostic information. Hence, the inclusion of PET results seems to improve the clinical counselling of patients with mRCC. Larger studies are needed to confirm these findings.

Nicotine relieves anxiogenic-like behavior in mice that overexpress the read-through variant of acetylcholinesterase but not in wild-type mice.

Stress increases vulnerability and causes relapse to drugs of abuse. The usually rare read-through variant of acetylcholinesterase (AChE-R) is causally involved in stress-related behaviors, and transgenic mice constitutively overexpressing AChE-R (TgR) show behaviors characteristic of chronic stress. We measured anxiety-like behavior on TgR and control mice under normal conditions and under long-term nicotine treatment. In addition, we measured epibatidine binding in the brain and transcription status in the striatum, using microarrays, in wild-type and TgR mice. TgR mice behaved as more anxious than controls, an effect normalized by long-term nicotine intake. In control mice, long-term nicotine augmented epibatidine binding in several areas of the brain, including the hippocampus and striatum. In TgR transgenics, long-term nicotine increased epibatidine binding in some areas but not in the hippocampus or the striatum. Because the striatum is involved in the mechanisms of drug addiction, we studied how the transgene affected striatal gene expression. Whole-genome DNA microarray showed that 23 transcripts were differentially expressed in TgR mouse striata, including 15 known genes, 7 of which are anxiety-related. Subsequent reverse-transcriptase polymerase chain reaction validated changes in 7 of those 15 genes, confirmed the increase trend in 5 more transcripts, and further revealed changes in 5 genes involved in cholinergic signaling. In summary, we found that nicotine acts as an anxiolytic in TgR mice but not in control mice and that continuously overexpressed AChE-R regulates striatal gene expression, modulating cholinergic signaling and stress-related pathways.

Modeling anaerobic digestion of dairy manure using the IWA Anaerobic Digestion Model no. 1 (ADM1).

The Anaerobic Digestion Model No. 1 (ADM1) can be used to describe treatment of dairy manure once manure characteristics have been incorporated in the model. In this paper a parameter set is presented that can be used with ADM1 for simulation of dairy manure digester performance. Model results have been verified with bench-scale experiments and reported data from full-scale systems. Model predictions fit experimental data best for biogas composition and digester effluent COD. Simulated biogas productions were inconsistent with measurements from three different digesters. The model overpredicted acetogenesis, resulting in higher simulated than observed acetate concentrations. However, total volatile acid concentrations were simulated reasonably well. The model consistently predicted higher inorganic nitrogen than measured or reported results, indicating a need for further research in that area. The presented model and associated parameter set can be used to simulate and optimize the performance of full-scale dairy manure digesters.

Catalytic carbonylation of functionalized diamines: application to the core structure of DMP 323 and DMP 450.

W(CO)(6)-catalyzed carbonylation provides an alternative to phosgene or phosgene derivatives such as 1,1-carbonyldiimidazole (CDI) for the conversion of amines to ureas. As an illustration, the core structure of the HIV protease inhibitors DMP 323 and DMP 450 has been prepared by catalytic carbonylation of diamine intermediates from the original syntheses.

Hydrogenation of carbon dioxide catalyzed by ruthenium trimethylphosphine complexes: the accelerating effect of certain alcohols and amines.

A trace amount of alcohol cocatalyst and a stoichiometric amount of base are required during the hydrogenation of CO(2) to formic acid catalyzed by ruthenium trimethylphosphine complexes. Variation of the choice of alcohol and base causes wide variation in the rate of reaction. Acidic, nonbulky alcohols and triflic acid increase the rate of hydrogenation an order of magnitude above that which can be obtained with traditionally used methanol or water. Similarly, use of DBU rather than NEt(3) increases the rate of reaction by an order of magnitude. Turnover frequencies up to 95,000 h(-1) have now been obtained, and even higher rates should be possible using the cocatalyst and amine combinations identified herein. Preliminary in situ NMR spectroscopic observations are described, and the possible roles of the alcohol and base are discussed.

Catalytic oxidative carbonylation of primary and secondary diamines to cyclic ureas. Optimization and substituent studies.

W(CO)(6)-catalyzed oxidative carbonylation of 1,3-propanediamine to the corresponding urea has been examined under a variety of conditions. Following optimization, the Thorpe-Ingold effect on ring closure was studied using 2,2-dialkyl-1,3-propanediamines. For the 2,2-dimethyl- and 2,2-dibutyl-1,3-propanediamines, the yields were increased significantly as compared to that of the unsubstituted case. The eight-membered cyclic urea 5-butyl-5-ethyl-1,3-diazepan-2-one (5f) was formed in 38% yield, while only trace amounts of the cyclic urea were produced from the parent 1,5-pentanediamine. In a study of secondary diamines, yields from the carbonylation of N,N'-dialkyl-2,2-dimethyl-1,3-propanediamines were lower than those obtained from the primary diamines. The main byproducts from secondary diamines were tetrahydropyrimidine derivatives formed from a competitive reaction of the substrate with the oxidant and base.

In situ formation of ruthenium catalysts for the homogeneous hydrogenation of carbon dioxide.

A total of 44 different phosphines were tested, in combination with [RuCl(2)(C(6)H(6))](2) and three other Ru(II) precursors, for their ability to form active catalysts for the hydrogenation of CO(2) to formic acid. Half (22) of the ligands formed catalysts of significant activity, and only 6 resulted in very high rates of production of formic acid. These were PMe(3), PPhMe(2), dppm, dppe, and cis- and trans-Ph(2)PCH=CHPPh(2). The in situ catalysts prepared from [RuCl(2)(C(6)H(6))](2) and any of these 6 phosphine ligands were found to be at least as efficient as the isolated catalyst RuCl(O(2)CMe)(PMe(3))(4). There was no correlation between the basicity of monophosphines (PR(3)) and the activity of the catalysts formed from them. However, weakly basic diphosphines formed highly active catalysts only if their bite angles were small, while more strongly basic diphosphines had the opposite trend. In situ (31)P NMR spectroscopy showed that trans-Ru(H)(2)(dppm)(2), trans-RuCl(2)(dppm)(2), trans-RuHCl(dppm)(2), cis-Ru(H)(O(2)CH)(dppm)(2), and cis-Ru(O(2)CH)(2)(dppm)(2) are produced as the major metal-containing species in reactions of dppm with [RuCl(2)(C(6)H(6))](2) under catalytic conditions at 50 degrees C.

Management by nurses of primary headache: a pilot study.

Primary headache is a very common condition and one that nurses encounter in many different care settings. Yet there is a lack of evidence as to whether advice given to sufferers is effective and what improvements may be expected in the condition. The aim of this study was to evaluate the advice given by a nurse to primary headache sufferers. The design was quasi-experimental. An experimental group (n = 19) and a control group (n = 25) of primary headache sufferers had their headache parameters of frequency, severity duration and disability (Migraine Disability Assessment) over the previous six months assessed. The experimental group then received advice in the form of health education from a nurse. Both groups kept a headache diary for six months. After six months both groups had their headache parameters assessed again. Compared to the control group, there was a significant reduction in the severity of headaches experienced by the experimental group (p = 0.006). Although there were reductions in frequency and duration of headaches experienced by the experimental group compared to the control group, these were not significant (p = 0.664 and p = 0.235, respectively). The Migraine Disability Assessment showed a trend towards reduced scores in the experimental group compared to controls which were not significant (p = 0.535). This pilot study suggests that health education can be effective in reducing the severity of headaches. However, a larger study over a longer period is needed to evaluate improvements in headache parameters.

Seasonal studies on commensal rats and their ectoparasites in a rural area of Egypt: the relationship of ectoparasites to the species, locality, and relative abundance of the host.

The present study was carried out in 3 villages, namely Kafr Ayoub Soliman, Kafr Ibrahim El-Aidi, and El-Sa'adat, Sharqiya Governorate, Egypt. A total of 519 rats was collected from the 3 study sites: 46.6% Rattus rattus, and 53.4% Rattus norvegicus. A total of 20,643 ectoparasites was recovered from R. rattus: 33.3% mites, 33.8% fleas, and 32.9% lice. From R. norvegicus a total of 40,997 ectoparasites was recovered: 28.9% mites, 31% fleas, and 40.1% lice. Three common mite species were recovered from both rat hosts, i.e., Ornithonyssus bacoti, Radfordia ensifera, and Laelaps nuttalli. Three common flea species were also recovered from both rat hosts, i.e., Echidnophaga gallinacea, Leptopsylla segnis, and Xenopsylla cheopis. Polyplax spinulosa was the only dominant louse species that infested both rat hosts. Rats did not show a definite breeding season, and the seasonal rat indices were generally low in different study sites. There were no significant differences between the prevalence of each of mites, fleas, and lice in both rat species. The total general indices of mites and fleas, on the other hand, was significantly higher in R. norvegicus. The general index of X. cheopis was high and ranged between 5.9 in R. rattus and 14.5 in R. norvegicus. Season-related changes were observed in the general index of each of L. segnis infesting both rat species and R. ensifera and O. bacoti infesting R. norvegicus. The prevalence and general indices of some ectoparasites showed differences related to the locality of their rat hosts. Seasonal changes in the general indices of some ectoparasites paralleled seasonal changes in the relative abundance of their rat hosts.

Effect of sex, size, and age of commensal rat hosts on the infestation parameters of their ectoparasites in a rural area of Egypt.

The present study was carried out in the Bilbeis area, Sharqiya Governorate, Egypt. A total of 127 male and 115 female black rats Rattus rattus and 124 male and 153 female Norway rats R. norvegicus was collected during the study period. A total of 20,643 and 40,997 ectoparasites was recovered from R. rattus and R. norvegicus, respectively. Three common mite species were recovered from both rat hosts: Ornithonyssus bacoti, Radfordia ensifera, and Laelaps nuttalli. Three common flea species were also recovered from both rat hosts: Echidnophaga gallinacea, Leptopsylla segnis, and Xenopsylla cheopis. Polyplax spinulosa was the only louse species that infested both rat hosts. Rats that lived more than 12 mo formed about 24 and 14% of the populations of the black and Norway rats, respectively. The prevalences and general indices of many ectoparasites were significantly higher on males than on females of both rat hosts. Seasonal differences in prevalences and general indices of many ectoparasites were recorded between the 2 sexes of both rat species. Prevalences and general indices of most ectoparasites on both rat species showed a tendency to increase with increasing size and age of rat hosts. The type and degree of correlation between prevalences and general indices on the one hand and each of body size and age on the other hand differed among the species of ectoparasites.

Electropalatography and the Linguagraph system.

This paper describes the technique of electropalatography and the development of Linguagraph, which is a user-friendly, clinical instrument, for measurement of tongue/palate contact, during speech. Linguagraph allows objective assessment of tongue function; appropriate targeting of therapy is therefore possible. Visual feedback is also provided, for therapy, and an objective measurement of outcome is easily obtained. Linguagraph was used, for both therapy and assessment, in a clinical trial. Technical aspects of Linguagraph and of the trial results are presented here. These suggest that the instrument will prove useful in the assessment and management of many speech disorders. Full clinical details of the trial are reported elsewhere.

The wavelength of light causing photophobia in migraine and tension-type headache between attacks.

OBJECTIVE: To ascertain the wavelength of light that patients with migraine and tension-type headache find uncomfortable between attacks. BACKGROUND: Photophobia is an abnormal perceptual sensitivity to light experienced by most patients with headache during and, also, between attacks. METHODS: We examined the discomfort threshold to light of low, medium, and high wavelengths in a group of patients with migraine (n=21), patients with tension-type headache (n=19), and healthy controls (n=21). RESULTS: The results indicate that the migraine group had significantly lower discomfort thresholds at the low (P=.001) and high (P=.031) wavelengths compared with both the tension-type headache and control groups; the latter two groups had similar average discomfort levels at these two wavelengths. With the medium wavelength, the control group had significantly higher discomfort thresholds than the migraine (P=.002) and tension-type headache (P=.031) groups; the latter two groups had similar discomfort levels at this wavelength. With unfiltered (white) light, the migraine group had the lowest discomfort threshold and the control group the highest (P=.026), whereas the tension-type headache group had an intermediate discomfort threshold. CONCLUSIONS: There were significant differences between migraineurs, patients with tension-type headache, and healthy controls in the wavelengths that are uncomfortable between attacks.

The impact of tick parasites on the behaviour of the lizard Tiliqua rugosa.

Populations of the Australian sleepy lizard, Tiliqua rugosa, near Mt. Mary, South Australia carry natural infestations of two tick species Aponomma hydrosauri and Amblyomma limbatum. In field experiments at two sites, 18 km apart, lizards with experimentally increased tick loads had smaller home ranges, moved shorter distances in a day, and were found basking more but moving less often than lizards from which ticks were experimentally removed. The results were consistent for adult lizards in two years, and for sub-adults in a third year. Laboratory trials showed that juvenile lizards that had tick infestations had lower sprint speeds than uninfested siblings, and that adults with tick infestations had less endurance than those that were uninfested. The results contrast with those of a previous survey that showed that lizards with high tick loads had greater body size and remained longer at a site, but indicate that there may be a balance, for lizards, between the fitness advantages in occupying habitats with high-quality resources, and the costs from parasites that also prefer those habitats.

An instrument for the multiparameter assessment of speech.

This paper describes the development of SNORS+, a clinical, user-friendly instrument for measurement of the articulators during speech. The design criteria for the instrument were based upon a wide-ranging review of current practice and available techniques. SNORS+ allows objective assessment of the function and co-ordination of key articulators. Appropriate targeting of therapy is therefore possible. Visual feedback is provided, for therapy, and an objective measurement of outcome is easily obtained. Preliminary results are presented. These suggest that the instrument will prove extremely useful in the assessment and management of many speech disorders.

Instrumental assessment and treatment of hypernasality, following maxillofacial surgery, using SNORS: a single case study.

The super nasal oral ratiometry system (SNORS) is a commercially available system which measures both nasal and oral airflow during speech, allowing the very rapid movement of the velum to be measured. SNORS uses a modified oxygen mask, which houses the airflow sensors and microphones, and a standard personal computer. By calculating nasalance (the percentage of airflow that is nasal), an estimation of velopharyngeal closure, which is independent of speech intensity, is achieved. SNORS can be used for objective assessment, where the subject is required to speak a number of words selected to demonstrate velopharyngeal function. SNORS also provides biofeedback, using a simple realtime display of nasal and oral airflow. Velopharyngeal insufficiency (VPI) is the inability to make adequate velpharyngeal closure, and may be the result of either neurological or, as in this case, structural abnormalities. It results in abnormal speech characteristics, such as omissions, substitutions or weak articulation of consonants, and hypernasality. T.W., a 52 year old male, had very hypernasal speech following extensive maxillofacial surgery, for the removal of a tonsillar carcinoma. SNORS was successfully used as both an assessment and a therapy tool in the treatment of this patient. The effectiveness of conventional speech and language therapy vs. SNORS biofeedback therapy was compared. Initially, while there was some movement of the velum, the patient could not achieve velopharyngeal closure. Conventional therapy aimed to strengthen and improve the function of the velum and following this there was some minimal improvement: the patient could now achieve, but not maintain, closure. Reassessment, following a non-treatment period, showed little further change. SNORS biofeedback therapy was then given. This raised the patient's awareness of his velopharyngeal function, thus helping him to maintain closure, thereby reducing hypernasality. SNORS therapy proved significantly more effective than conventional speech and language therapy, in this case. Further intervention is outlined, and the benefits of multiparameter assessment of speech are discussed.

An electrochemical, kinetic, and spectroscopic characterization of [2Fe-2S] vegetative and heterocyst ferredoxins from Anabaena 7120 with mutations in the cluster binding loop.

Residues within the cluster binding loops of plant-type [2Fe-2S] ferredoxins are highly conserved and serve to structurally stabilize this unique region of the protein. We have investigated the influence of these residues on the thermodynamic reduction potentials and rate constants of electron transfer to ferredoxin:NADP+ reductase (FNR) by characterizing various single and multiple site-specific mutants of both the vegetative (VFd) and the heterocyst (HFd) [2Fe-2S] ferredoxins from Anabaena. Incorporation of residues from one isoform into the polypeptide backbone of the other created hybrid mutants whose reduction potentials either were not significantly altered or were shifted, but did not reconcile the 33-mV potential difference between VFd and HFd. The reduction potential of VFd appears relatively insensitive to mutations in the binding loop, excepting nonconservative variations at position 78 (T78A/I) which resulted in approximately 40- to 50-mV positive shifts compared to wild type. These perturbations may be linked to the role of the T78 side chain in stabilizing an ordered water channel between the iron-sulfur cluster and the surface of the wild-type protein. While no thermodynamic barrier to electron transfer to FNR is created by these potential shifts, the electron-transfer reactivities of mutants T78A/I (as well as T48A which has a wild-type-like potential) are reduced to approximately 55-75% that of wild type. These studies suggest that residues 48 and 78 are involved in the pathway of electron transfer between VFd and FNR and/or that mutations at these positions induce a unique, but unproductive orientation of the two proteins within the protein-protein complex.