Bacterial sensors define intracellular free energies for correct enzyme metalation.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

On the Antibacterial Activity of Azacarboxylate Ligands: Lowered Metal Ion Affinities for Bis-amide Derivatives of EDTA do not mean Reduced Activity.

EDTA is widely used as an inhibitor of bacterial growth, affecting the uptake and control of metal ions by microorganisms. We describe the synthesis and characterisation of two symmetrical bis-amide derivatives of EDTA, featuring glycyl or pyridyl substituents: AmGly2 and AmPy2 . Metal ion affinities (logK) have been evaluated for a range of metals (Mg2+ , Ca2+ , Fe3+ , Mn2+ , Zn2+ ), revealing less avid binding compared to EDTA. The solid-state structures of AmGly2 and of its Mg2+ complex have been determined crystallographically. The latter shows an unusual 7-coordinate, capped octahedral Mg2+ centre. The antibacterial activities of the two ligands and of EDTA have been evaluated against a range of health-relevant bacterial species, three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and a Gram positive (Staphylococcus aureus). The AmPy2 ligand is the only one that displays a significant inhibitory effect against K. pneumoniae, but is less effective against the other organisms. AmGly2 exhibits a more powerful inhibitory effect against E. coli at lower concentrations than EDTA (<3 mm) or AmPy2 , but loses its efficacy at higher concentrations. The growth inhibition of EDTA and AmGly2 on mutant E. coli strains with defects in outer-membrane lipopolysaccharide (LPS) structures has been assessed to provide insight into the unexpected behaviour. Taken together, the results contradict the assumption of a simple link between metal ion affinity and antimicrobial efficacy.

Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.

Alternative protein sources: science powered startups to fuel food innovation.

Harnessing the potential of considerable food security efforts requires the ability to translate them into commercial applications. This is particularly true for alternative protein sources and startups being on the forefront of innovation represent the latest advancements in this field.

Impact of continuous low level heatwrap therapy in acute low back pain patients: subjective and objective measurements.

OBJECTIVES: Muscular pain is usually associated with increased muscle tension resulting in a vicious tension-pain-cycle, leading to increased alertness and stress. However, this has not been broadly evaluated using objective methods, for example, looking at neurophysiologic changes. The focus of this study was, therefore, to combine objective [spontaneous electroencephalogram (EEG) as a surrogate of alertness and stress] with subjective parameters (self-assessed pain affected variables) to investigate the effect of continuous low-level heat therapy in low back pain (LBP)-patients. METHODS: This investigation was a randomized, active controlled, parallel-designed study. Thirty patients were randomly assigned to one of 2 groups: the control group, in which patients were provided with oral analgesics (nonsteroidal anti-inflammatory drug) and instructed to use it if needed, and the treatment group, in which patients in addition to oral analgesics as rescue medication were provided with a heatwrap therapy. The objective parameters were assessed by measuring the power of frequency bands in the spontaneous EEG. The subjective parameters (sleep pattern, well-being, pain intensity, etc.) were assessed by a Pain, Sleep, and Stress Questionnaire. RESULTS: In the EEG-recordings, the heatwrap therapy group showed decreased power in Beta-1 and Beta-2 frequency bands compared with the control group, indicating a reduction in arousal. Also, in comparison to the control group, the heatwrap therapy group reported significantly reduced LBP, everyday situations being less stressful, a better night's sleep, and a decreased number of daytime naps. DISCUSSION: In addition to classic psychophysical assessment of pain-related parameters and sleep quality, performance in daily life, we were able to obtain objective measures (EEG) that suggest an acute therapeutic relaxation on the basis of the central nervous system effects accompanying the reported significant pain relief. We believe that this was due to a reduced nociceptive information load in LBP-patients after the use of the heatwrap therapy.

Fine control of metal concentrations is necessary for cells to discern zinc from cobalt.

Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but metal binding is not specific in vitro. Here we show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity of Zur, ZntR, RcnR and FrmR in Salmonella Typhimurium. In cells, at non-inhibitory elevated concentrations, Zur and ZntR, only respond to Zn(II), RcnR to cobalt and FrmR to formaldehyde. However, in vitro all these sensors bind non-cognate metals, which alters DNA binding. We model the responses of these sensors to intracellular-buffered concentrations of Co(II) and Zn(II) based upon determined abundances, metal affinities and DNA affinities of each apo- and metalated sensor. The cognate sensors are modelled to respond at the lowest concentrations of their cognate metal, explaining specificity. However, other sensors are modelled to respond at concentrations only slightly higher, and cobalt or Zn(II) shock triggers mal-responses that match these predictions. Thus, perfect metal specificity is fine-tuned to a narrow range of buffered intracellular metal concentrations.

The virtue of innovation: innovation through the lenses of biological evolution.

We rehearse the processes of innovation and discovery in general terms, using as our main metaphor the biological concept of an evolutionary fitness landscape. Incremental and disruptive innovations are seen, respectively, as successful searches carried out locally or more widely. They may also be understood as reflecting evolution by mutation (incremental) versus recombination (disruptive). We also bring a platonic view, focusing on virtue and memory. We use 'virtue' as a measure of efforts, including the knowledge required to come up with disruptive and incremental innovations, and 'memory' as a measure of their lifespan, i.e. how long they are remembered. Fostering innovation, in the evolutionary metaphor, means providing the wherewithal to promote novelty, good objective functions that one is trying to optimize, and means to improve one's knowledge of, and ability to navigate, the landscape one is searching. Recombination necessarily implies multi- or inter-disciplinarity. These principles are generic to all kinds of creativity, novel ideas formation and the development of new products and technologies.

Product and technology innovation: what can biomimicry inspire?

Biomimicry (bio- meaning life in Greek, and -mimesis, meaning to copy) is a growing field that seeks to interpolate natural biological mechanisms and structures into a wide range of applications. The rise of interest in biomimicry in recent years has provided a fertile ground for innovation. This review provides an eco-system based analysis of biomimicry inspired technology and product innovation. A multi-disciplinary framework has been developed to accomplish this analysis and the findings focus on the areas that have been most strikingly affected by the application of biomimicry and also highlight the emerging trends and opportunity areas.

Cost-effectiveness of low-level heat wrap therapy for low back pain.

OBJECTIVES: To evaluate the cost-effectiveness and budget impact of a new heat wrap therapy for low back pain compared to paracetamol and ibuprofen from the perspective of the UK National Health Service (NHS). METHODS: We evaluated cost-effectiveness using data from a phase III trial comparing the three therapies in 371 patients aged 18 to 55 years presenting with acute uncomplicated low back pain. The primary effectiveness measure used was successful treatment, defined as both clinically meaningful pain relief and clinically meaningful reduction in disability. We conducted a simple evaluation using NHS prescription costs and a modeled extrapolation including the costs of further treatment and consultations for patients treated unsuccessfully or with adverse events. Uncertainty was addressed using nonparametric bootstrapping and sensitivity analyses. RESULTS: Successful treatment was reported by 57% of patients treated with heat wrap therapy, 26% treated with paracetamol and 18% treated with ibuprofen (P < 0.05 for heat wrap vs. both other groups). NHS prescription cost per patient was estimated to be 1.35 pounds Sterling for heat wrap therapy, 0.26 pounds Sterling for paracetamol, and 0.28 pounds Sterling for ibuprofen and cost per successful treatment was 3.52 pounds Sterling for heat wrap therapy compared to paracetamol, and 2.72 pounds Sterling compared to ibuprofen. In the modeled extrapolation, NHS cost per patient was 27.77 pounds Sterling for heat wrap therapy, 34.20 pounds Sterling for paracetamol, and 36.04 pounds Sterling for ibuprofen. Sensitivity analyses indicated that the findings were robust to plausible changes in data and assumptions. CONCLUSIONS: Economic evaluation of this study suggests that the NHS cost of introducing heat wrap therapy in place of oral analgesics would be modest and heat wrap therapy might potentially reduce the total cost of managing episodes of lower back pain.