Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

Background The thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools. Results Here we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype. Conclusions The procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing

Pion irradiation studies of CVD diamond detectors

We report here the results of a test to ascertain the radiation hardness properties of CVD diamond detectors to 300 MeV/c pions. In this test, CVD diamond detectors were exposed to 8 \times 10^{13} pions per cm^2 using the high intensity pion beam at the Paul Scherrer Institut. For comparison, silicon photodiodes were exposed to similar fluences at the same time. The measurements and the dosimetry during the irradiations are described herein. As expected, the silicon devices degraded. The diamond devices showed no degradation in collected charge and no increase in leakage current

Long-Stay Psychiatric Patients: A Prospective Study Revealing Persistent Antipsychotic-Induced Movement Disorder

OBJECTIVE: The purpose of this study was to assess the frequency of persistent drug-induced movement disorders namely, tardive dyskinesia (TD), parkinsonism, akathisia and tardive dystonia in a representative sample of long-stay patients with chronic severe mental illness. METHOD: Naturalistic study of 209, mainly white, antipsychotic-treated patients, mostly diagnosed with psychotic disorder. Of this group, the same rater examined 194 patients at least two times over a 4-year period, with a mean follow-up time of 1.1 years, with validated scales for TD, parkinsonism, akathisia, and tardive dystonia. RESULTS: The frequencies of persistent movement disorders in the sample were 28.4% for TD, 56.2% for parkinsonism, 4.6% for akathisia and 5.7% for tardive dystonia. Two-thirds of the participants displayed at least one type of persistent movement disorder. CONCLUSIONS: Persistent movement disorder continues to be the norm for long-stay patients with chronic mental illness and long-term antipsychotic treatment. Measures are required to remedy this situation

Development of CVD diamond radiation detectors

Diamond is a nearly ideal material for detecting ionizing radiation. Its outstanding radiation hardness, fast charge collection and low leakage current allow a diamond detector to be used in high ra diation, high temperature and in aggressive chemical media. We have constructed charged particle detectors using high quality CVD diamond. Characterization of the diamond samples and various detect ors are presented in terms of collection distance, $d=\mu E \tau$, the average distance electron-hole pairs move apart under the influence of an electric field, where $\mu$ is the sum of carrier mo bilities, $E$ is the applied electric field, and $\tau$ is the mobility weighted carrier lifetime. Over the last two years the collection distance increased from $\sim$ 75 $\mu$m to over 200 $\mu$ m. With this high quality CVD diamond a series of micro-strip and pixel particle detectors have been constructed. These devices were tested to determine their position resolution and signal to n oise performance. Diamond detectors were exposed to large fluences of pions, protons and neutrons to establish their radiation hardness properties. The results of these tests and their correlati on with the characterization studies are presented

Proton Irradiation of CVD Diamond Detectors for High Luminosity Experiments at the LHC

CVD diamond shows promising properties for use as a position sensitive detector for experiments in the highest radiation areas at the Large Hadron Collider. In order to study the radiation hardn ess of diamond we exposed CVD diamond detector samples to 24~GeV/$c$ and 500~MeV protons up to a fluence of $5\times 10^{15}~p/{\rm cm^2}$. We measured the charge collection distance, the ave rage distance electron hole pairs move apart in an external electric field, and leakage currents before, during, and after irradiation. The charge collection distance remains unchanged up to $1\ times 10^{15}~p/{\rm cm^2}$ and decreases by $\approx$40~\% at $5\times 10^{15}~p/{\rm cm^2}$. Leakage currents of diamond samples were below 1~pA before and after irradiation. The particle indu ced currents during irradiation correlate well with the proton flux. In contrast to diamond, a silicon diode, which was irradiated for comparison, shows the known large increase in leakage curren t. We conclude that CVD diamond detectors are radiation hard to 24~GeV/$c$ and 500~MeV protons up to at least $1\times 10^{15}~p/{\rm cm^2}$ without signal loss

Gene expression of NMDA receptor subunits in the cerebellum of elderly patients with schizophrenia

To determine if NMDA receptor alterations are present in the cerebellum in schizophrenia, we measured NMDA receptor binding and gene expression of the NMDA receptor subunits in a post-mortem study of elderly patients with schizophrenia and non-affected subjects. Furthermore, we assessed influence of genetic variation in the candidate gene neuregulin-1 (NRG1) on the expression of the NMDA receptor in an exploratory study. Post-mortem samples from the cerebellar cortex of ten schizophrenic patients were compared with nine normal subjects. We investigated NMDA receptor binding by receptor autoradiography and gene expression of the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D by in situ hybridization. For the genetic study, we genotyped the NRG1 polymorphism rs35753505 (SNP8NRG221533). Additionally, we treated rats with the antipsychotics haloperidol or clozapine and assessed cerebellar NMDA receptor binding and gene expression of subunits to examine the effects of antipsychotic treatment. Gene expression of the NR2D subunit was increased in the right cerebellum of schizophrenic patients compared to controls. Individuals carrying at least one C allele of rs35753505 (SNP8NRG221533) showed decreased expression of the NR2C subunit in the right cerebellum, compared to individuals homozygous for the T allele. Correlation with medication parameters and the animal model revealed no treatment effects. In conclusion, increased NR2D expression results in a hyperexcitable NMDA receptor suggesting an adaptive effect due to receptor hypofunction. The decreased NR2C expression in NRG1 risk variant may cause a deficit in NMDA receptor function. This supports the hypothesis of an abnormal glutamatergic neurotransmission in the right cerebellum in the pathophysiology of schizophrenia

The effects of cognitive therapy versus 'treatment as usual' in patients with major depressive disorder

BACKGROUND: Major depressive disorder afflicts an estimated 17% of individuals during their lifetimes at tremendous suffering and costs. Cognitive therapy may be an effective treatment option for major depressive disorder, but the effects have only had limited assessment in systematic reviews. METHODS/PRINCIPAL FINDINGS: Cochrane systematic review methodology, with meta-analyses and trial sequential analyses of randomized trials, are comparing the effects of cognitive therapy versus 'treatment as usual' for major depressive disorder. To be included the participants had to be older than 17 years with a primary diagnosis of major depressive disorder. Altogether, we included eight trials randomizing a total of 719 participants. All eight trials had high risk of bias. Four trials reported data on the 17-item Hamilton Rating Scale for Depression and four trials reported data on the Beck Depression Inventory. Meta-analysis on the data from the Hamilton Rating Scale for Depression showed that cognitive therapy compared with 'treatment as usual' significantly reduced depressive symptoms (mean difference -2.15 (95% confidence interval -3.70 to -0.60; P<0.007, no heterogeneity)). However, meta-analysis with both fixed-effect and random-effects model on the data from the Beck Depression Inventory (mean difference with both models -1.57 (95% CL -4.30 to 1.16; P = 0.26, I(2) = 0) could not confirm the Hamilton Rating Scale for Depression results. Furthermore, trial sequential analysis on both the data from Hamilton Rating Scale for Depression and Becks Depression Inventory showed that insufficient data have been obtained. DISCUSSION: Cognitive therapy might not be an effective treatment for major depressive disorder compared with 'treatment as usual'. The possible treatment effect measured on the Hamilton Rating Scale for Depression is relatively small. More randomized trials with low risk of bias, increased sample sizes, and broader more clinically relevant outcomes are needed