Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.

A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae

Identification of Rhizoctonia solani, R. oryzae and R. oryzae-sativae, components of the rice sheath disease complex, is extremely difficult and often inaccurate and as a result may hinder the success of extensive breeding programmes throughout Asia. In this study, primers designed from unique regions within the rDNA internal transcribed spacers have been used to develop a rapid PCR-based diagnostic test to provide an accurate identification of the species on rice. Tests on the specificity of the primers concerned showed that they provide the means for accurate identification of the Rhizoctonia species responsible for sheath diseases in rice.

Idiosyncratic species effects confound size-based predictions of responses to climate change

Understanding and predicting the consequences of warming for complex ecosystems and indeed individual species remains a major ecological challenge. Here, we investigated the effect of increased seawater temperatures on the metabolic and consumption rates of five distinct marine species. The experimental species reflected different trophic positions within a typical benthic East Atlantic food web, and included a herbivorous gastropod, a scavenging decapod, a predatory echinoderm, a decapod and a benthic-feeding fish. We examined the metabolism-body mass and consumption-body mass scaling for each species, and assessed changes in their consumption efficiencies. Our results indicate that body mass and temperature effects on metabolism were inconsistent across species and that some species were unable to meet metabolic demand at higher temperatures, thus highlighting the vulnerability of individual species to warming. While body size explains a large proportion of the variation in species' physiological responses to warming, it is clear that idiosyncratic species responses, irrespective of body size, complicate predictions of population and ecosystem level response to future scenarios of climate change. © 2012 The Royal Society.

Interacting kinetics of neutral and ionic species in an atmospheric-pressure helium-oxygen plasma with humid air impurities

We unravel the complex chemistry in both the neutral and ionic systems of a radio-frequency-driven atmospheric-pressure plasma in a helium-oxygen mixture (He-0.5% O) with air impurity levels from 0 to 500 ppm of relative humidity from 0% to 100% using a zero-dimensional, time-dependent global model. Effects of humid air impurity on absolute densities and the dominant production and destruction pathways of biologically relevant reactive neutral species are clarified. A few hundred ppm of air impurity crucially changes the plasma from a simple oxygen-dependent plasma to a complex oxygen-nitrogen-hydrogen plasma. The density of reactive oxygen species decreases from 10 to 10 cm, which in turn results in a decrease in the overall chemical reactivity. Reactive nitrogen species (10 cm ), atomic hydrogen and hydroxyl radicals (10-10 cm) are generated in the plasma. With 500 ppm of humid air impurity, the densities of positively charged ions and negatively charged ions slightly increase and the electron density slightly decreases (to the order of 10 cm). The electronegativity increases up to 2.3 compared with 1.5 without air admixture. Atomic hydrogen, hydroxyl radicals and oxygen ions significantly contribute to the production and destruction of reactive oxygen and reactive nitrogen species. © 2013 IOP Publishing Ltd.

Molecular mechanism and physiological role of active-deactive transition of mitochondrial complex I

The unique feature ofmitochondrial complex I is the so-called A/D transition (active-deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~10 min) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1-10 min) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys of mitochondrially encoded subunit ND3 makes the Dform susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III + IV (S) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

Complex organic molecules in protoplanetary disks

Context. Protoplanetary disks are vital objects in star and planet formation, possessing all the material, gas and dust, which may form a planetary system orbiting the new star. Small, simple molecules have traditionally been detected in protoplanetary disks; however, in the ALMA era, we expect the molecular inventory of protoplanetary disks to significantly increase.

Aims. We investigate the synthesis of complex organic molecules (COMs) in protoplanetary disks to put constraints on the achievable chemical complexity and to predict species and transitions which may be observable with ALMA.

Methods. We have coupled a 2D steady-state physical model of a protoplanetary disk around a typical T Tauri star with a large gas-grain chemical network including COMs. We compare the resulting column densities with those derived from observations and perform ray-tracing calculations to predict line spectra. We compare the synthesised line intensities with current observations and determine those COMs which may be observable in nearby objects. We also compare the predicted grain-surface abundances with those derived from cometary comae observations.

Results. We find COMs are efficiently formed in the disk midplane via grain-surface chemical reactions, reaching peak grain-surface fractional abundances similar to 10(-6)-10(-4) that of the H nuclei number density. COMs formed on grain surfaces are returned to the gas phase via non-thermal desorption; however, gas-phase species reach lower fractional abundances than their grain-surface equivalents, similar to 10(-12)-10(-7). Including the irradiation of grain mantle material helps build further complexity in the ice through the replenishment of grain-surface radicals which take part in further grain-surface reactions. There is reasonable agreement with several line transitions of H2CO observed towards T Tauri star-disk systems. There is poor agreement with HC3(N) lines observed towards LkCa 15 and GO Tau and we discuss possible explanations for these discrepancies. The synthesised line intensities for CH3OH are consistent with upper limits determined towards all sources. Our models suggest CH3OH should be readily observable in nearby protoplanetary disks with ALMA; however, detection of more complex species may prove challenging, even with ALMA "Full Science" capabilities. Our grain-surface abundances are consistent with those derived from cometary comae observations providing additional evidence for the hypothesis that comets (and other planetesimals) formed via the coagulation of icy grains in the Sun's natal disk.

Evaluation of methodologies to determine vegetable oil species present in oil mixtures: Proposition of an approach to meet the EU legislation demands for correct vegetable oils labelling

Refined vegetable oils are widely used in the food industry as ingredients or components in many processed food products in the form of oil blends. To date, the generic term 'vegetable oil' has been used in the labelling of food containing oil blends. With the introduction of new EU Regulation for Food Information (1169/2011) due to take effect in 2014, the oil species used must be clearly identified on the package and there is a need for development of fit for purpose methodology for industry and regulators alike to verify the oil species present in a product. The available methodologies that may be employed to authenticate the botanical origin of a vegetable oil admixture were reviewed and evaluated. The majority of the sources however, described techniques applied to crude vegetable oils such as olive oil due to the lack of refined vegetable oil focused studies. Nevertheless, DNA based typing methods and stable isotopes procedures were found not suitable for this particular purpose due to several issues. Only a small number of specific chromatographic and spectroscopic fingerprinting methods in either targeted or untargeted mode were found to be applicable in potentially providing a solution to this complex authenticity problem. Applied as a single method in isolation, these techniques would be able to give limited information on the oils identity as signals obtained for various oil types may well be overlapping. Therefore, more complex and combined approaches are likely to be needed to identify the oil species present in oil blends employing a stepwise approach in combination with advanced chemometrics. Options to provide such a methodology are outlined in the current study.

A necessarily complex model to explain the biogeography of the amphibians and reptiles of Madagascar

Pattern and process are inextricably linked in biogeographic analyses, though we can observe pattern, we must infer process. Inferences of process are often based on ad hoc comparisons using a single spatial predictor. Here, we present an alternative approach that uses mixed-spatial models to measure the predictive potential of combinations of hypotheses. Biodiversity patterns are estimated from 8,362 occurrence records from 745 species of Malagasy amphibians and reptiles. By incorporating 18 spatially explicit predictions of 12 major biogeographic hypotheses, we show that mixed models greatly improve our ability to explain the observed biodiversity patterns. We conclude that patterns are influenced by a combination of diversification processes rather than by a single predominant mechanism. A ‘one-size-fits-all’ model does not exist. By developing a novel method for examining and synthesizing spatial parameters such as species richness, endemism and community similarity, we demonstrate the potential of these analyses for understanding the diversification history of Madagascar’s biota.

Disinfection of Burkholderia cepacia complex from non-touch taps in a neonatal nursery

Burkholderia cepacia complex (Bcc) comprises nine closely related species or genomovars. It is an important causative agent of opportunistic infections and waterborne nosocomial infections. B. cepacia (formerly genomovar I) was identified from the blood culture of a baby in our neonatal unit (NU) in March 2005. B. cepacia was isolated four times from clinical specimens since the introduction of non-touch taps in the NU from 2000 to 2005 and only once from 1994 to 2000. Environmental samples were collected from the NU, including tap water from non-touch taps. Clinical and environmental isolates of Bcc were characterized using molecular identification and strain typing. A literature review was undertaken to delineate a method for eradication of Bcc. Several variations for hot water eradication of the organism from the taps were attempted. Genotyping and molecular analysis revealed that tap water isolates were B. cenocepacia which was a different species from the B. cepacia isolated from blood cultures of the neonate. However, B. cenocepacia has been known to cause nosocomial outbreaks and it was eventually eradicated from the NU by using repeated thermal shock (hot water at 65 degrees C for 10 min), changing taps and decolonizing sinks with hypochlorite. Molecular typing is useful in assisting the investigation of Bcc nosocomial infections.

Burkholderia cepacia complex epidemiology in persons with cystic fibrosis from Australia and New Zealand

The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.

Spatio-temporal Change in Crowned (Propithecus coronatus) and Decken’s Sifaka (Propithecus deckenii) Habitat in the Mahavavy-Kinkony Wetland Complex, Madagascar.

The crowned sifaka (Propithecus coronatus) and Decken’s sifaka (Propithecus deckenii) are Endangered lemurs endemic to west and central Madagascar. Both have suffered habitat loss and fragmentation throughout their ranges. The goal
of this study, conducted in the Mahavavy-Kinkony Wetland Complex (MKWC) in northwestern Madagascar, was to assess the effects of historical change in the species’ habitats, and to model the potential impact of further land-use change on their habitats. The IDRISI Andes Geographical Information System and image-processing software was used for satellite-image classifiation, and the Land Change Modeler was used to compare the natural habitat of the species from 1973 to 2005, and to predict available habitat for 2050. We analyzed two forests in the MKWC occupied by P. coronatus (Antsilaiza and Anjohibe), and three forests occupied by P. deckenii (Tsiombikibo, Marofandroboka and Andohaomby). The two forests occupied by P. coronatus contracted during the period 1949–1973, but then expanded to exceed their 1949 area by 28% in 2005. However, the land change model predicted that they will contract again to match their 1949 area by 2050, and will again lose their corridor connection, meaning that the conservation gains for this species in the complex are at risk of being reversed. The three forests occupied by P. deckenii have declined in area steadily since 1949, losing 20% of their original area by 2005, and are predicted to lose a further 15% of their original area by 2050. Both species are therefore at risk of becoming even more threatened if land-use change continues within the complex. Improved conservation of the remaining forest is recommended to avoid further loss, as well as ecological restoration and reforestation to promote connectivity between the forests. A new strategy for controlling agriculture and forest use is required in order to avoid further destruction of the forest.

Further evidence for cryptic north-western refugia in Europe? Mitochondrial phylogeography of the sibling species Pipistrellus pipistrellus and Pipistrellus pygmaeus

The geographic ranges of European plants and animals underwent periods of contraction and re-colonisation during the climatic oscillations of the Pleistocene. The southern Mediterranean peninsulas (Iberian, Italian and Balkan) have been considered the most likely refugia for temperate/warm adapted species. Recent studies however have revealed the existence of extra-Mediterranean refugia, including the existence of cryptic north-west European refugia during the Last Glacial Maxima (24-14.6 kyr BP). In this study we elucidated the phylogeographic history of two sibling bat species, Pipistrellus pipistrellus and P. pygmaeus in their western European range. We sequenced the highly variable mtDNA D-loop for 167 samples of P. pipistrellus (n = 99) and P. pygmaeus (n = 68) and combined our data with published sequences from 331 individuals. Using phylogenetic methodologies we assessed their biogeographic history. Our data support a single eastern European origin for populations of P. pygmaeus s.str., yet multiple splits and origins for populations of P. pipistrellus s.str., including evidence for refugia within refugia and potential cryptic refugia in north western Europe and in the Caucasus. This complex pattern in the distribution of mtDNA haplotypes supports a long history for P. pipistrellus s.str. in Europe, and the hypothesis that species with a broad ecological niche may have adapted and survived outside southern peninsula throughout the LGM.

Intracellular survival of Burkholderia cepacia complex in phagocytic cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance, and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils and dendritic cells, combined with advances to genetically manipulate these bacteria have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of B. cenocepacia within macrophages.