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+.

Catabolite repression of the citST two-component system in Bacillus subtilis

In Bacillus subtilis, expression of the citrate transporter CitM is under strict control. Transcription of the citM gene is induced by citrate in the medium mediated by the CitS-CitT two-component system and repressed by rapidly degraded carbon sources mediated by carbon catabolite repression (CCR). In this study, we demonstrate that citST genes are part of a bicistronic operon. The promoter region was localized in a stretch of 58 base pairs upstream of the citS gene by deletion experiments. Transcription of the operon was repressed in the presence of glucose by the general transcription factor CcpA. A distal consensus cre site in the citS-coding sequence was implicated in the mechanism of repression. Furthermore, this repression was relieved in Bacillus subtilis mutants deficient in CcpA or Hpr/Crh, components essential to CCR. Thus, we demonstrate that CCR represses the expression of the citST operon, which is responsible for the induction of citM, through the cre site located 1326 bp from transcriptional start site of citST

Weissella halotolerans W22 combines arginine deiminase and ornithine decarboxylation pathways and converts arginine to putrescine

Aims: To demonstrate that the meat food strain Weissella halotolerans combines an ornithine decarboxylation pathway and an arginine deiminase (ADI) pathway and is able to produce putrescine, a biogenic amine. Evidence is shown that these two pathways produce a proton motive force (PMF). Methods and Results: Internal pH in W. halotolerans was measured with the sensitive probe 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein. Membrane potential was measured with the fluorescent probe 3,3'-dipropylthiocarbocyanine iodine. Arginine and ornithine transport studies were made under several conditions, using cells loaded or not loaded with the biogenic amine putrescine. ADI pathway caused an increase in Delta pH dependent on the activity of F(0)F(1)ATPase. Ornithine decarboxylation pathway generates both a Delta pH and a Delta Psi. Both these pathways lead to the generation of a PMF. Conclusions: Weissella halotolerans W22 combines an ADI pathway and an ornithine decarboxylation pathway, conducing to the production of the biogenic amine putrescine and of a PMF. Transport studies suggest the existence of a unique antiporter arginine/putrescine in this lactic acid bacteria strain. Significance and Impact of the Study: The coexistence of two different types of amino acid catabolic pathways, leading to the formation of a PMF, is shown for a Weissella strain for the first time. Moreover, a unique antiport arginine/putrescine is hypothesized to be present in this food strain

Recommendations for the use of next-generation sequencing (NGS) for patients with metastatic cancers: a report from the ESMO Precision Medicine Working Group

Next-generation sequencing (NGS) allows sequencing of a high number of nucleotides in a short time frame at an affordable cost. While this technology has been widely implemented, there are no recommendations from scientific societies about its use in oncology practice. The European Society for Medical Oncology (ESMO) is proposing three levels of recommendations for the use of NGS. Based on the current evidence, ESMO recommends routine use of NGS on tumour samples in advanced non-squamous non-small-cell lung cancer (NSCLC), prostate cancers, ovarian cancers and cholangiocarcinoma. In these tumours, large multigene panels could be used if they add acceptable extra cost compared with small panels. In colon cancers, NGS could be an alternative to PCR. In addition, based on the KN158 trial and considering that patients with endometrial and small-cell lung cancers should have broad access to anti-programmed cell death 1 (anti-PD1) antibodies, it is recommended to test tumour mutational burden (TMB) in cervical cancers, well- and moderately-differentiated neuroendocrine tumours, salivary cancers, thyroid cancers and vulvar cancers, as TMB-high predicted response to pembrolizumab in these cancers. Outside the indications of multigene panels, and considering that the use of large panels of genes could lead to few clinically meaningful responders, ESMO acknowledges that a patient and a doctor could decide together to order a large panel of genes, pending no extra cost for the public health care system and if the patient is informed about the low likelihood of benefit. ESMO recommends that the use of off-label drugs matched to genomics is done only if an access programme and a procedure of decision has been developed at the national or regional level. Finally, ESMO recommends that clinical research centres develop multigene sequencing as a tool to screen patients eligible for clinical trials and to accelerate drug development, and prospectively capture the data that could further inform how to optimise the use of this technology

Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material

Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5%concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detectio

Application of circulating tumor DNA in prospective clinical oncology trials - standardization of preanalytical conditions

Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive, and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various preanalytical conditions on the quality of cell-free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant. The following preanalytical conditions were investigated: (a) different time intervals to plasma isolation (1, 24, and 96 h) and (b) different preservatives in blood collection tubes (EDTA, CellSave, and BCT). The quality of cfDNA/ctDNA was assessed by DNA quantification, digital polymerase chain reaction (dPCR) for somatic variant detection and a β-actin fragmentation assay for DNA contamination from lysed leukocytes. In 11 (69%) of our 16 patients, we were able to detect the known somatic variant in ctDNA. We observed a time-dependent increase in cfDNA concentrations in EDTA tubes, which was positively correlated with an increase in wild-type copy numbers and large DNA fragments (> 420 bp). Using different preserva

Structural genomics target selection for the New York consortium on membrane protein structure

The New York Consortium on Membrane Protein Structure (NYCOMPS), a part of the Protein Structure Initiative (PSI) in the USA, has as its mission to establish a high-throughput pipeline for determination of novel integral membrane protein structures. Here we describe our current target selection protocol, which applies structural genomics approaches informed by the collective experience of our team of investigators. We first extract all annotated proteins from our reagent genomes, i.e. the 96 fully sequenced prokaryotic genomes from which we clone DNA. We filter this initial pool of sequences and obtain a list of valid targets. NYCOMPS defines valid targets as those that, among other features, have at least two predicted transmembrane helices, no predicted long disordered regions and, except for community nominated targets, no significant sequence similarity in the predicted transmembrane region to any known protein structure. Proteins that feed our experimental pipeline are selected by defining a protein seed and searching the set of all valid targets for proteins that are likely to have a transmembrane region structurally similar to that of the seed. We require sequence similarity aligning at least half of the predicted transmembrane region of seed and target. Seeds are selected according to their feasibility and/or biological interest, and they include both centrally selected targets and community nominated targets. As of December 2008, over 6,000 targets have been selected and are currently being processed by the experimental pipeline. We discuss how our target list may impact structural coverage of the membrane protein space