Purification and Reconstitution of the Glutamate Carrier GltT of the Thermophilic Bacterium Bacillus stearothermophilus

An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5α grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost, suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

Three Poems: The Dog at the Hospital; Bracken Ferns; Branta Canadensis

These three poems reflect the speaker\u27s refugee experience and his adjustment to the new land and the natural world and present an account of his love, companionship, and memory of war

Melanoma NOS1 expression promotes dysfunctional IFN signaling.

In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells

Peptide exchange on MHC-I by TAPBPR is driven by a negative allostery release cycle.

Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex

Independent position correction on tumor and lymph nodes; consequences for bladder cancer irradiation with two combined IMRT plans

Abstract Background The application of lipiodol injections as markers around bladder tumors combined with the use of CBCT for image guidance enables daily on-line position correction based on the position of the bladder tumor. However, this might introduce the risk of underdosing the pelvic lymph nodes. In this study several correction strategies were compared. Methods For this study set-up errors and tumor displacements for ten complete treatments were generated; both were based on the data of 10 bladder cancer patients. Besides, two IMRT plans were made for 20 patients, one for the elective field and a boost plan for the tumor. For each patient 10 complete treatments were simulated. For each treatment the dose was calculated without position correction (option 1), correction on bony anatomy (option 2), on tumor only (option 3) and separately on bone for the elective field (option 4). For each method we analyzed the D99% for the tumor, bladder and lymph nodes and the V95% for the small intestines, rectum, healthy part of the bladder and femoral heads. Results CTV coverage was significantly lower with options 1 and 2. With option 3 the tumor coverage was not significantly different from the treatment plan. The ΔD99% (D99%, option n - D99%, treatment plan) for option 4 was small, but significant. For the lymph nodes the results from option 1 differed not significantly from the treatment plan. The median ΔD99% of the other options were small, but significant. ΔD99% for PTVbladder was small for options 1, 2 and 4, but decreased up to -8.5 Gy when option 3 was applied. Option 4 is the only method where the difference with the treatment plan never exceeds 2 Gy. The V95% for the rectum, femoral heads and small intestines was small in the treatment plan and this remained so after applying the correction options, indicating that no additional hot spots occurred. Conclusions Applying independent position correction on bone for the elective field and on tumor for the boost separately gives on average the best target coverage, without introducing additional hot spots in the healthy tissue.</p

Local Dose Effects for Late Gastrointestinal Toxicity After Hypofractionated and Conventionally Fractionated Modern Radiotherapy for Prostate Cancer in the HYPRO Trial

Purpose: Late gastrointestinal (GI) toxicity after radiotherapy for prostate cancer may have significant impact on the cancer survivor's quality of life. To da

Spatio-temporal Evolution of Diesel Sprays at the Early Start of Injection

The impact of injector life on the spatio-temporal evolution of fuel spray quality was optically investigated using high speed imaging techniques. Both new and used injectors, which had been used in intense operation for up to 90,000 miles, were considered in this investigation. Used injectors are prone to wear, deposit formation, and altered internal nozzle flow. High resolution SEM images clearly portray the presence of carbonaceous deposits both at the injector tip as well as within the holes of used injectors. Investigations revealed that used injectors tend to produce a chaotic hole-to-hole variation at the start of each injection, resulting in an asymmetric early fuel spraypenetration pattern in the first 500μs. Often those sprays thatsuffereda reduced spray tippenetration rate at the start of injection also showed off-axis transient expansions, and those sprays appeared to be bulky compared to other sprays. Following the early asymmetric spray penetration phase of injection the retarded sprays undergo rapid acceleration with time, and this transformed the early asymmetric spray pattern into a nearly uniform spray pattern from all the orifices in the quasi-steady state regime. The hole-to-hole penetration variations and the resultant asymmetric spray structure at the early start are therefore short lived transient phenomena. However if the radial expansion of the spray is large during the early phase, the radially expanded plume remains almost at the same radial location due to lack of local axial momentum, even after different time instants of spray tip propagation. This appears as a bulge to the spray and can eventually end up as a stationary local pocket of fuel vapor close to the nozzle for the entire duration of injection. This may alter the ignition, flame lift-off and entrainment characteristics of sprays injected from used or deposit rich injectors

Investigation of Spray Angle Measurement Techniques

The in-cylinder fluid-dynamic processes of fuel injection and air entrainment influence the structure and shape of evolving fuel sprays, which can subsequently alter the ignition, combustion and pollutant formation processes in diesel engines. Different spray angle detection methods have been used in the literature to investigate the global and local spray characteristics. In this work, the five most widely used diesel spray angle detection methods were identified, and used to evaluate the characteristic features of each detection method: methods with a detection range based on the spray penetration length, methods with a fixed detection range in the near and far-field spray region, triangular-based methods, and methods based on averaging local data points. The sprays were acquired from our spray chamber and processed with different thresholding techniques to explore the differences between spray angle detection methods. All five methods generated a similar global trend of spray angle variation for temporally evolving sprays over the complete injection period. However, the actual spray angle values detected by each method were not always comparable. The differences in spray angle values between the different detection methods were larger during the early start of injection, and these differences systematically decreased as the spray approached a steady-state. The methods that detected the angle in the far-field demonstrated lower spatiotemporal variability when compared to the methods that detected the angle in the near-field. An assessment ofthecomparabilitybetweenangledetectionmethodswasmadeandtheoutcomeprovidesguidanceforthe selection of the spray angle detection method.Engineering and Physical Sciences Research Council in the UK (EPSRC