Generation of <em>Escherichia coli</em> nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in ∼5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be ∼5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 μM than was the WT enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy

Can type 2 diabetes and its associated complications be prevented or delayed in people with intermediate hyperglycaemia?

Diabetes affects one in 11 adults in the WHO European Region. It is a key risk factor for cardiovascular diseases, kidney failure, vision loss and nerve damage. Intermediate hyperglycaemia is a state in which blood glucose levels are above the normal range but below the threshold for diabetes. It is associated with an increased risk for type 2 diabetes, obesity, cardiovascular diseases and mortality. This review assessed the effects of interventions for people with intermediate hyperglycaemia. Results from randomized controlled trials indicate that the risk of developing type 2 diabetes in people with intermediate hyperglycaemia is reduced by lifestyle and (some) pharmacological interventions. Most of the available evidence did not find a difference in mortality or other serious health outcomes for either pharmacological or lifestyle interventions. However, the follow-up periods may have been too short for health outcomes to have emerged. The current evidence suggests that the risk of developing type 2 diabetes is reduced through intervention at the point of intermediate hyperglycaemia, but that the effects of these interventions on long-term health outcomes are unclear

CACNA1C hypermethylation is associated with bipolar disorder

The CACNA1C gene, encoding a subunit of the L-type voltage-gated calcium channel is one of the best-supported susceptibility genes for bipolar disorder (BD). Genome-wide association studies have identified a cluster of non-coding single-nucleotide polymorphisms (SNPs) in intron 3 to be highly associated with BD and schizophrenia. The mechanism by which these SNPs confer risk of BD appears to be through an altered regulation of CACNA1C expression. The role of CACNA1C DNA methylation in BD has not yet been addressed. The aim of this study was to investigate if CACNA1C DNA methylation is altered in BD. First, the methylation status of five CpG islands (CGIs) across CACNA1C in blood from BD subjects (n=40) and healthy controls (n=38) was determined. Four islands were almost completely methylated or completely unmethylated, while one island (CGI 3) in intron 3 displayed intermediate methylation levels. In the main analysis, the methylation status of CGI 3 was analyzed in a larger sample of BD subjects (n=582) and control individuals (n=319). Out of six CpG sites that were investigated, five sites showed significant hypermethylation in cases (lowest P=1.16 × 10(-7) for CpG35). Nearby SNPs were found to influence the methylation level, and we identified rs2238056 in intron 3 as the strongest methylation quantitative trait locus (P=2.6 × 10(-7)) for CpG35. In addition, we found an increased methylation in females, and no difference between bipolar I and II. In conclusion, we find that CACNA1C methylation is associated with BD and suggest that the regulatory effect of the non-coding risk variants involves a shift in DNA methylation

What is the effect of population-level screening of asymptomatic adults for type 2 diabetes mellitus or intermediate hyperglycaemia on health outcomes?

Diabetes mellitus is one of the world's fastest growing chronic conditions. It is associated with heart disease, eye and kidney problems, and premature death. Intermediate hyperglycaemia, a state in which blood glucose levels are above the normal range but below the threshold for diabetes, is associated with an increased risk for type 2 diabetes (T2DM), obesity, cardiovascular diseases and mortality. The review assessed whether population-level screening for intermediate hyperglycaemia and T2DM can improve health outcomes. A single, underpowered, biased study found no benefit of population-level screening for T2DM to reduce morbidity or mortality. No studies reported whether treatment after screen detection improved health outcomes compared with either no treatment or treatment after later symptomatic detection. One underpowered study found no significant difference in health outcomes between more- and less-intensive treatment after screen detection. In summary, there is currently no evidence that screening for T2DM or IHG reduces morbidity or mortality

Can type 2 diabetes and its associated complications be prevented or delayed in people with intermediate hyperglycaemia?

Diabetes affects one in 11 adults in the WHO European Region. It is a key risk factor for cardiovascular diseases, kidney failure, vision loss and nerve damage. Intermediate hyperglycaemia is a state in which blood glucose levels are above the normal range but below the threshold for diabetes. It is associated with an increased risk for type 2 diabetes, obesity, cardiovascular diseases and mortality. This review assessed the effects of interventions for people with intermediate hyperglycaemia. Results from randomized controlled trials indicate that the risk of developing type 2 diabetes in people with intermediate hyperglycaemia is reduced by lifestyle and (some) pharmacological interventions. Most of the available evidence did not find a difference in mortality or other serious health outcomes for either pharmacological or lifestyle interventions. However, the follow-up periods may have been too short for health outcomes to have emerged. The current evidence suggests that the risk of developing type 2 diabetes is reduced through intervention at the point of intermediate hyperglycaemia, but that the effects of these interventions on long-term health outcomes are unclear

Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair. © 2014 The Author(s)

A Rapid Gamma-Ray Glow Flux Reduction Observed From 20 km Altitude

Two gamma-ray glows were observed by a high-altitude NASA ER-2 aircraft flying at 20 km altitude over a thunderstorm in Colorado, USA. The flux of the first glow rapidly intensified and then abruptly decreased within a few tens of milliseconds. On a timescale of seconds, the flux decrease occurred simultaneously with a hybrid intra-cloud/cloud-to-ground lightning discharge beneath the aircraft. However, a more detailed analysis of the discharge dynamics indicated that the discharge activity was unusually calm during the actual period of the flux decrease. The lightning was observed with on-board antennas, optical sensor, and ground-based lightning mapping and location networks. Its closest activity was 12 km away from the aircraft, below and slightly ahead the course. The gamma-ray flux reduction happened roughly in the middle of the lightning development process. The glow spectral analysis for the periods of a weak and strong flux enhancement has been done. The spectra were found to be background-like and similar to each other.publishedVersio

Gamma Ray Glow Observations at 20-km Altitude

In the spring of 2017 an ER‐2 aircraft campaign was undertaken over continental United States to observe energetic radiation from thunderstorms and lightning. The payload consisted of a suite of instruments designed to detect optical signals, electric fields, and gamma rays from lightning. Starting from Georgia, USA, 16 flights were performed, for a total of about 70 flight hours at a cruise altitude of 20 km. Of these, 45 flight hours were over thunderstorm regions. An analysis of two gamma ray glow events that were observed over Colorado at 21:47 UT on 8 May 2017 is presented. We explore the charge structure of the cloud system, as well as possible mechanisms that can produce the gamma ray glows. The thundercloud system we passed during the gamma ray glow observation had strong convection in the core of the cloud system. Electric field measurements combined with radar and radio measurements suggest an inverted charge structure, with an upper negative charge layer and a lower positive charge layer. Based on modeling results, we were not able to unambiguously determine the production mechanism. Possible mechanisms are either an enhancement of cosmic background locally (above or below 20 km) by an electric field below the local threshold or an enhancement of the cosmic background inside the cloud but then with normal polarity and an electric field well above the Relativistic Runaway Electron Avalanche threshold.publishedVersio

Effects of an isoenergetic low Glycaemic Index (GI) diet on liver fat accumulation and gut microbiota composition in patients with non-alcoholic fatty liver disease (NAFLD): A study protocol of an efficacy mechanism evaluation

Introduction A Low Glycaemic Index (LGI) diet is a proposed lifestyle intervention in non-alcoholic fatty liver diseases (NAFLD) which is designed to reduce circulating blood glucose levels, hepatic glucose influx, insulin resistance and de novo lipogenesis. A significant reduction in liver fat content through following a 1-week LGI diet has been reported in healthy volunteers. Changes in dietary fat and carbohydrates have also been shown to alter gut microbiota composition and lead to hepatic steatosis through the gut-liver axis. There are no available trials examining the effects of an LGI diet on liver fat accumulation in patients with NAFLD; nor has the impact of consuming an LGI diet on gut microbiota composition been studied in this population. The aim of this trial is to investigate the effects of LGI diet consumption on liver fat content and its effects on gut microbiota composition in participants with NAFLD compared with a High Glycaemic Index (HGI) control diet. Methods and analysis A 2×2 cross-over randomised mechanistic dietary trial will allocate 16 participants with NAFLD to a 2-week either HGI or LGI diet followed by a 4-week wash-out period and then the LGI or HGI diet, alternative to that followed in the first 2 weeks. Baseline and postintervention (four visits) outcome measures will be collected to assess liver fat content (using MRI/S and controlled attenuation parameter-FibroScan), gut microbiota composition (using 16S RNA analysis) and blood biomarkers including glycaemic, insulinaemic, liver, lipid and haematological profiles, gut hormones levels and short-chain fatty acids. Ethics and dissemination Study protocol has been approved by the ethics committees of The University of Nottingham and East Midlands Nottingham-2 Research Ethics Committee (REC reference 19/EM/0291). Data from this trial will be used as part of a Philosophy Doctorate thesis. Publications will be in peer-reviewed journals. Trial registration number NCT04415632