Characterization of the active site and calcium binding in cytochromecnitrite reductases

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction

Light-Driven H2 Evolution and C═C or C═O Bond Hydrogenation by Shewanella oneidensis : A Versatile Strategy for Photocatalysis by Nonphotosynthetic Microorganisms

Photocatalytic chemical synthesis by coupling abiotic photosensitizers to purified enzymes provides an effective way to overcome the low conversion efficiencies of natural photosynthesis while exploiting the high catalytic rates and selectivity of enzymes as renewable, earth-abundant electrocatalysts. However, the selective synthesis of multiple products requires more versatile approaches and should avoid the time-consuming and costly processes of enzyme purification. Here we demonstrate a cell-based strategy supporting light-driven H2 evolution or the hydrogenation of C═C and C═O bonds in a nonphotosynthetic microorganism. Methylviologen shuttles photoenergized electrons from water-soluble photosensitizers to enzymes that catalyze H2 evolution and the reduction of fumarate, pyruvate, and CO2 in Shewanella oneidensis. The predominant reaction is selected by the experimental conditions, and the results allow rational development of cell-based strategies to harness nature’s intrinsic catalytic diversity for selective light-driven synthesis of a wide range of products

Outcome of a Simple Novel Technique to Reduce Soft Tissue Complications in Open Tendoachilles Injury: A Series of 20 Patients

Introduction: Open tendoachilles injuries are rare and associated with significant soft tissues complications. The objective of the present study was to assess the clinical outcome and safety of a simple and minimally invasive technique, with a goal to assess if it may help minimise flap and wound related complications in open tendoachilles injuries. Materials and methods: This prospective study of four years duration included 20 patients with open tendoachilles injuries managed with a simple minimally invasive tunnel technique. The primary outcome variable was occurrence of a major soft tissue complication. The secondary outcome variables included functional outcome measured using AOFAS Ankle hind foot score, re-rupture of tendoachilles and need for revision surgery. Results: None of the patients in the present series developed a serious soft tissue complication. Based upon the AOFAS hind foot scoring system, good to excellent outcome was achieved in 19 (95%) patients. All the patients were able to perform tip toe walking at six months post-surgery. None of the patients had a re-rupture of the tendoachilles and no patient needed a revision surgery. The complications encountered include thickening of the tendon at the repair site (15%), superficial wound infection (5%), stitch granuloma (5%) and hypertrophic scar (5%). Conclusion: This technique seems to be promising in reducing the soft tissue complications associated with the surgical management of open tendoachilles injuries. Most patients had a good final clinical outcome. The technique is safe, simple and reproducible. However, further randomised control studies with a larger sample size assessing the technique are recommended

GLAST: Understanding the High Energy Gamma-Ray Sky

We discuss the ability of the GLAST Large Area Telescope (LAT) to identify, resolve, and study the high energy gamma-ray sky. Compared to previous instruments the telescope will have greatly improved sensitivity and ability to localize gamma-ray point sources. The ability to resolve the location and identity of EGRET unidentified sources is described. We summarize the current knowledge of the high energy gamma-ray sky and discuss the astrophysics of known and some prospective classes of gamma-ray emitters. In addition, we also describe the potential of GLAST to resolve old puzzles and to discover new classes of sources.Comment: To appear in Cosmic Gamma Ray Sources, Kluwer ASSL Series, Edited by K.S. Cheng and G.E. Romer

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV–vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme.

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-μs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

SummaryBackground The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors—the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57·8% (95% CI 56·6–58·8) of global deaths and 41·2% (39·8–42·8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211·8 million [192·7 million to 231·1 million] global DALYs), smoking (148·6 million [134·2 million to 163·1 million]), high fasting plasma glucose (143·1 million [125·1 million to 163·5 million]), high BMI (120·1 million [83·8 million to 158·4 million]), childhood undernutrition (113·3 million [103·9 million to 123·4 million]), ambient particulate matter (103·1 million [90·8 million to 115·1 million]), high total cholesterol (88·7 million [74·6 million to 105·7 million]), household air pollution (85·6 million [66·7 million to 106·1 million]), alcohol use (85·0 million [77·2 million to 93·0 million]), and diets high in sodium (83·0 million [49·3 million to 127·5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Funding Bill & Melinda Gates Foundation

Tight Junction-Related Barrier Contributes to the Electrophysiological Asymmetry across Vocal Fold Epithelium

Electrophysiological homeostasis is indispensable to vocal fold hydration. We investigate tight junction (TJ)-associated components, occludin and ZO-1, and permeability with or without the challenge of a permeability-augmenting agent, histamine. Freshly excised ovine larynges are obtained from a local abattoir. TJ markers are explored via reverse transcriptase polymerase chain reaction (RT-PCR). Paracellular permeabilities are measured in an Ussing system. The gene expression of both TJ markers is detected in native ovine vocal fold epithelium. Luminal histamine treatment significantly decreases transepithelial resistance (TER) (N = 72, p<0.01) and increases penetration of protein tracer (N = 35, p<0.001), respectively, in a time-, and dose-dependent fashion. The present study demonstrates that histamine compromises TJ-related paracellular barrier across vocal fold epithelium. The detection of TJ markers indicates the existence of typical TJ components in non-keratinized, stratified vocal fold epithelium. The responsiveness of paracellular permeabilities to histamine would highlight the functional significance of this TJ-equivalent system to the electrophysiological homeostasis, which, in turn, regulates the vocal fold superficial hydration

A whole-cell biosensor for the detection of gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).Carla M. Zammit, Davide Quaranta, Shane Gibson, Anita J. Zaitouna, Christine Ta, Joël Brugger, Rebecca Y. Lai, Gregor Grass, Frank Reit