42 research outputs found
Spectroscopic and electrochemical studies of di-heme thiosulphate dehydrogenases
Thiosulphate (SSO32-) and tetrathionate (S4O62-) are found in environments where numerous bacterial species live. Some of these bacteria express di-heme thiosulphate dehydrogenase (TsdA) enzymes that catalyse the interconversion of these sulphur compounds:
2 SSO32- ↔ S4O62- + 2 e- (EM = +198 mV)
Electrons originate or terminate in this redox couple during respiration or photosynthesis in these bacteria. This is likely to give them a niche competitive advantage against other organisms lacking this enzyme in environments where these compounds are present. The catalytic site of TsdA enzymes is heme I which has the unusual axial ligand pairing His/Cys-. Heme II is an electron transfer site which has His/Met ligation in most TsdA enzymes (eg. Campylobacter jejuni (Cj) TsdA), but which has a redox-driven switch between His/Lys and His/Met ligation in two known TsdA enzymes (eg. Allochromatium vinosum (Av) TsdA).
This work uses magnetic circular dichroism (MCD) to identify the heme ligation states for recombinant TsdA enzymes Cj TsdA and Av TsdA in solution and several variants where these ligands are substituted. Protein film electrochemistry (PFE) also characterised the electroactivity of the TsdA hemes in these proteins. This, in combination with electrochemically-poised MCD samples, was used to determine the mid-point potentials of native and variant hemes. Additionaly these techniques confirmed the Av TsdA heme II ligand switch and permitted investigation of its kinetics. Remarkably, the number of electrons transferred by Cj TsdA heme I in PFE is different to the number transferred by heme II. In comparison, the number of electrons transferred by the Av TsdA hemes are equal, although introducing Cj TsdA-like heme II ligation, introduces unequal electron transfer. Replacing the heme I cysteine ligand with methionine in Cj TsdA causes both hemes to have equal electron transfer. Liquid chromatography mass spectrometry (LC-MS)-resolved native covalent adducts to the heme I cysteine ligand, which modulate the electroactivity of heme I.
A catalytic mechanism proposed for TsdA enzymes features covalent modification of the heme I cysteine ligand. Chemical treatments, substrates and substrate analogues have been used to change these modifications in a predictable manner, allowing for the characterisation of TsdA with defined heme I cysteine modifications. Combining data from PFE, MCD and LC-MS the previously-proposed catalytic mechanism is expanded further. Evidence suggests that the substrate-augmented site has adducts to the heme I cysteine which also control electron transfer to the TsdA hemes. This electron transfer is not adequately explained by conventional theory of redox centres in proteins suggesting that the reality in TsdA enzymes is more complex than this typically assumed model
Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzyme
Thiosulfate dehydrogenases (TsdA) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic circular dichroism (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a micro-aerobe human pathogen, and from the purple sulfur bacterium Allochromatium vinosum. In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase respectively. The active site Heme 1 in both enzymes has His/Cys− ligation in the ferric and ferrous states and the midpoint potentials (Em) of the corresponding redox transformations are similar, −185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em, −129 mV) to His/Met (Em,+266 mV) but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s)
His/Met heme ligation in the PioA outer membrane cytochrome enabling light-driven extracellular electron transfer by Rhodopseudomonas palustris TIE-1
A growing number of bacterial species are known to move electrons across their cell envelopes. Naturally this occurs in support of energy conservation and carbon-fixation. For biotechnology it allows electron exchange between bacteria and electrodes in microbial fuel cells and during microbial electrosynthesis. In this context Rhodopseudomonas palustris TIE-1 is of much interest. These bacteria respond to light by taking electrons from their external environment, including electrodes, to drive CO2-fixation. The PioA cytochrome, that spans the bacterial outer membrane, is essential for this electron transfer and yet little is known about its structure and electron transfer properties. Here we reveal the ten c-type hemes of PioA are redox active across the window +250 to -400 mV versus Standard Hydrogen Electrode and that the hemes with most positive reduction potentials have His/Met and His/H2O ligation. These chemical and redox properties distinguish PioA from the more widely studied family of MtrA outer membrane decaheme cytochromes with ten His/His ligated hemes. We predict a structure for PioA in which the hemes form a chain spanning the longest dimension of the protein, from Heme 1 to Heme 10. Hemes 2, 3 and 7 are identified as those most likely to have His/Met and/or His/H2O ligation. Sequence analysis suggests His/Met ligation of Heme 2 and/or 7 is a defining feature of decaheme PioA homologs from over 30 different bacterial genera. His/Met ligation of Heme 3 appears to be less common and primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class
Extrafloral nectaries in Opuntia robusta
Descubrimos la existencia de nectáreos extraflorales en Opuntia robustaTo our knowledge, there are no studies about the structure and ecological function of extra- floral nectaries (EFNs) in Opuntia robusta. This is the first description of EFNs in O. robusta, where young spines have an interesting structure and a secreting function, which are differ- ent from EFNs described in other Cactaceae species. We used light, scanning-electron, and transmission-electron microscopy to examine morphology, anatomy, and ultrastructure of the secretory spines in areoles in female and hermaphrodite individuals of O. robusta. Young cladodes develop areoles with modified and secretory spines as EFNs only active during the early growth phase. EFNs are non-vascularized structures, with no stomata, that consist of a basal meristematic tissue, a middle elongation region, and an apical secretory cone formed by large globular epidermal cells, containing nectar and medullar elongated cells. We observed the presence of Golgi apparatus, vesicles and plastids in the medullar and sup-epidermal cells of the spine. We propose that the nectar is stored in the globular cells at the apex of the spine and secreted by breaking through the globular cells or by pores. We recorded a more frequent presence of ants on younger cladode sprouts producing young secreting spines: this result is parallel with the predictions of Optimal Defense Hypothesis, which states that younger plant organs should be better defended than older ones because their loss produces a higher fitness impairment. Although Diaz-Castelazo’s hypothesis states that a more complex structure of EFNs correlates with their lower among-organs dispersion, comparing to less complex EFNs, non-vascularized structure of EFNs in O. robusta is not associated with their higher among-organs dispersion likened to O. stricta, which produces vascularized EFNs. We provide evidence that this characteristic is not a good taxonomic fea- ture of Opuntia genus. Moreover, the comparison of EFNs of O. robusta and O. stricta sug- gests that the hypothesis of Diaz-Castelazo should be revised: it is rather a rule but not a law.CONACyT 180694/201
N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson\u27s Disease: Preliminary Clinical and Cell Line Data.
BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson\u27s disease (PD).
METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson\u27s Disease Rating Scale (UPDRS) to measure clinical symptoms.
RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p
CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted.
TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651
Act now against new NHS competition regulations: an open letter to the BMA and the Academy of Medical Royal Colleges calls on them to make a joint public statement of opposition to the amended section 75 regulations.
Resolution of inflammation: a new therapeutic frontier
Dysregulated inflammation is a central pathological process in diverse disease states. Traditionally, therapeutic approaches have sought to modulate the pro- or anti-inflammatory limbs of inflammation, with mixed success. However, insight into the pathways by which inflammation is resolved has highlighted novel opportunities to pharmacologically manipulate these processes — a strategy that might represent a complementary (and perhaps even superior) therapeutic approach. This Review discusses the state of the art in the biology of resolution of inflammation, highlighting the opportunities and challenges for translational research in this field
Laparoscopy in management of appendicitis in high-, middle-, and low-income countries: a multicenter, prospective, cohort study.
BACKGROUND: Appendicitis is the most common abdominal surgical emergency worldwide. Differences between high- and low-income settings in the availability of laparoscopic appendectomy, alternative management choices, and outcomes are poorly described. The aim was to identify variation in surgical management and outcomes of appendicitis within low-, middle-, and high-Human Development Index (HDI) countries worldwide. METHODS: This is a multicenter, international prospective cohort study. Consecutive sampling of patients undergoing emergency appendectomy over 6 months was conducted. Follow-up lasted 30 days. RESULTS: 4546 patients from 52 countries underwent appendectomy (2499 high-, 1540 middle-, and 507 low-HDI groups). Surgical site infection (SSI) rates were higher in low-HDI (OR 2.57, 95% CI 1.33-4.99, p = 0.005) but not middle-HDI countries (OR 1.38, 95% CI 0.76-2.52, p = 0.291), compared with high-HDI countries after adjustment. A laparoscopic approach was common in high-HDI countries (1693/2499, 67.7%), but infrequent in low-HDI (41/507, 8.1%) and middle-HDI (132/1540, 8.6%) groups. After accounting for case-mix, laparoscopy was still associated with fewer overall complications (OR 0.55, 95% CI 0.42-0.71, p < 0.001) and SSIs (OR 0.22, 95% CI 0.14-0.33, p < 0.001). In propensity-score matched groups within low-/middle-HDI countries, laparoscopy was still associated with fewer overall complications (OR 0.23 95% CI 0.11-0.44) and SSI (OR 0.21 95% CI 0.09-0.45). CONCLUSION: A laparoscopic approach is associated with better outcomes and availability appears to differ by country HDI. Despite the profound clinical, operational, and financial barriers to its widespread introduction, laparoscopy could significantly improve outcomes for patients in low-resource environments. TRIAL REGISTRATION: NCT02179112
Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy
Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe
Mortality of emergency abdominal surgery in high-, middle- and low-income countries
Background: Surgical mortality data are collected routinely in high-income countries, yet virtually no low- or middle-income countries have outcome surveillance in place. The aim was prospectively to collect worldwide mortality data following emergency abdominal surgery, comparing findings across countries with a low, middle or high Human Development Index (HDI).
Methods: This was a prospective, multicentre, cohort study. Self-selected hospitals performing emergency surgery submitted prespecified data for consecutive patients from at least one 2-week interval during July to December 2014. Postoperative mortality was analysed by hierarchical multivariable logistic regression.
Results: Data were obtained for 10 745 patients from 357 centres in 58 countries; 6538 were from high-, 2889 from middle- and 1318 from low-HDI settings. The overall mortality rate was 1⋅6 per cent at 24 h (high 1⋅1 per cent, middle 1⋅9 per cent, low 3⋅4 per cent; P < 0⋅001), increasing to 5⋅4 per cent by 30 days (high 4⋅5 per cent, middle 6⋅0 per cent, low 8⋅6 per cent; P < 0⋅001). Of the 578 patients who died, 404 (69⋅9 per cent) did so between 24 h and 30 days following surgery (high 74⋅2 per cent, middle 68⋅8 per cent, low 60⋅5 per cent). After adjustment, 30-day mortality remained higher in middle-income (odds ratio (OR) 2⋅78, 95 per cent c.i. 1⋅84 to 4⋅20) and low-income (OR 2⋅97, 1⋅84 to 4⋅81) countries. Surgical safety checklist use was less frequent in low- and middle-income countries, but when used was associated with reduced mortality at 30 days.
Conclusion: Mortality is three times higher in low- compared with high-HDI countries even when adjusted for prognostic factors. Patient safety factors may have an important role. Registration number: NCT02179112 (http://www.clinicaltrials.gov)