A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo.

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and as a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo

A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo

Protection against cardiac ischemia-reperfusion injury by hypothermia and by inhibition of succinate accumulation and oxidation is additive.

Hypothermia induced at the onset of ischemia is a potent experimental cardioprotective strategy for myocardial infarction. The aim of our study was to determine whether the beneficial effects of hypothermia may be due to decreasing mitochondria-mediated mechanisms of damage that contribute to the pathophysiology of ischemia/reperfusion injury. New Zealand male rabbits were submitted to 30 min of myocardial ischemia with hypothermia (32 °C) induced by total liquid ventilation (TLV). Hypothermia was applied during ischemia alone (TLV group), during ischemia and reperfusion (TLV-IR group) and normothermia (Control group). In all the cases, ischemia was performed by surgical ligation of the left anterior descending coronary artery and was followed by 3 h of reperfusion before assessment of infarct size. In a parallel study, male C57BL6/J mice underwent 30 min myocardial ischemia followed by reperfusion under either normothermia (37 °C) or conventionally induced hypothermia (32 °C). In both the models, the levels of the citric acid cycle intermediate succinate, mitochondrial complex I activity were assessed at various times. The benefit of hypothermia during ischemia on infarct size was compared to inhibition of succinate accumulation and oxidation by the complex II inhibitor malonate, applied as the pro-drug dimethyl malonate under either normothermic or hypothermic conditions. Hypothermia during ischemia was cardioprotective, even when followed by normothermic reperfusion. Hypothermia during ischemia only, or during both, ischemia and reperfusion, significantly reduced infarct size (2.8 ± 0.6%, 24.2 ± 3.0% and 49.6 ± 2.6% of the area at risk, for TLV-IR, TLV and Control groups, respectively). The significant reduction of infarct size by hypothermia was neither associated with a decrease in ischemic myocardial succinate accumulation, nor with a change in its rate of oxidation at reperfusion. Similarly, dimethyl malonate infusion and hypothermia during ischemia additively reduced infarct size (4.8 ± 2.2% of risk zone) as compared to either strategy alone. Hypothermic cardioprotection is neither dependent on the inhibition of succinate accumulation during ischemia, nor of its rapid oxidation at reperfusion. The additive effect of hypothermia and dimethyl malonate on infarct size shows that they are protective by distinct mechanisms and also suggests that combining these different therapeutic approaches could further protect against ischemia/reperfusion injury during acute myocardial infarction

A mouse model reproducing the pathophysiology of neonatal group B streptococcal infection

Group B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.The authors gratefully acknowledge the help of Encarnaca̧ ̃o Ribeiro for excellent technical assistance, Joana Tavares for assisting with IVIS Lumina LT, Susana Roque for the luminex instrument experiments, the Molecular Microbiology group at i3S for microscope use, and the Portuguese architect and artist Gil Ferreira da Silva for the artworks included in the last figure. This work was supported by funds from Foundation for Science and Technology (FCT), European Regional Development Fund (FEDER) and Compete under project POCI-01-0145-FEDER-016607 (PTDC/IMI-MIC/1049/2014) and from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). T.S. and A.M. were supported by Investigador FCT (IF/00875/2012 and IF/00753/2014), POPH and Fundo Social Europeu. E.B.A. and C.C.P. hold postdoctoral fellowships from FCT (PTDC/IMI-MIC/1049/2014 and SFRH/BPD/91962/2012). Ar.F. and P.T.C. were supported by Laboratoire d’Excellence (LABEX) Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID).info:eu-repo/semantics/publishedVersio

Improved community detection for WDN partitioning in the dual topology based on segments and valves

This paper proposes a formulation of modularity tailored to the dual water distribution network (WDN) topology based on segments and valves, to be conveniently adopted for the partitioning into district-metered areas (DMAs). Notably, it allows considering both properties to be made uniform across DMAs, such as water demand or total pipe length, and properties to be made uniform inside each DMA, such as nodal ground elevations or pipe age for the sake of pressure regulation or maintenance easiness, respectively. This paper also proposes a new algorithm for the identification of the optimal clustering of WDN segments into any desired number of DMAs. Taking as a starting point any WDN clustering solution, i.e., the solution obtained with Newman’s fast algorithm for community detection, the novel algorithm operates by exploring changes in the community of belonging to segments lying in the boundary between adjacent communities, by applying an optimization inspired by the simulated annealing technique. The applications of the novel modularity formulation and optimization algorithm to two case studies yield well-performing clustering solutions in terms of engineering judgment criteria, such as the low number of inter-DMA boundary pipes, uniformity of DMAs and hydraulic performance

General Method Based on Regressive Relationships to Parameterize the Three-Parameter Depth–Duration–Frequency Curve

This paper aims to present simple regressive equations to estimate the parameters of the three-parameter depth–duration–frequency (DDF) curve (3p-DDF), which accurately expresses, for a preassigned return period, the relationship between the rainfall depth and the rainfall duration over large duration ranges, from below 1 h (i.e., tens of minutes) to above 1 h (up to 24 h). These equations are developed to relate their parameters to those of the two-parameter DDF curve (2p-DDF), which can be estimated more easily being based on more readily available data related to rainfall durations above 1 h. In the applications, the regressive equations are first calibrated using recent pluviographic data in northern Italy, Germany, and Sweden. Two validation steps are then carried out to test the equations in terms of estimated rainfall depths using the same data as those used in the calibration step and data of stations from other geographic areas, i.e., Sicily in southern Italy, and from the past century, respectively. The results obtained prove this methodology capable of providing reliable estimation of short-duration rainfalls with various return periods in the absence of measurements with fine temporal resolution

Vancomycin is protective in a neonatal mouse model of Staphylococcus epidermidis-potentiated hypoxic-ischemic brain injury

Infection is correlated with increased risk of neurodevelopmental sequelae in preterm infants. In modeling neonatal brain injury, Toll-like receptor agonists have often been used to mimic infections and induce inflammation. Using the most common cause of bacteremia in preterm infants, Staphylococcus epidermidis, we present a more clinically relevant neonatal mouse model that addresses the combined effects of bacterial infection together with subsequent hypoxic-ischemic brain insult. Currently, there is no neuroprotective treatment for the preterm population. Hence, we tested the neuroprotective effects of vancomycin with and without adjunct therapy using the anti-inflammatory agent pentoxifylline. We characterized the effects of S. epidermidis infection on the inflammatory response in the periphery and the brain, as well as the physiological changes in the central nervous system that might affect neurodevelopmental outcomes. Intraperitoneal injection of postnatal day 4 mice with a live clinical isolate of S. epidermidis led to bacteremia and induction of proinflammatory cytokines in the blood, as well as transient elevations of neutrophil and monocyte chemotactic cytokines and caspase 3 activity in the brain. When hypoxia-ischemia was induced postinfection, more severe brain damage was observed in infected animals than in saline-injected controls. This infection-induced inflammation and potentiated brain injury was inoculum dose dependent and was alleviated by the antibiotic vancomycin. Pentoxifylline did not provide any additional neuroprotective effect. Thus, we show for the first time that live S. epidermidis potentiates hypoxic-ischemic preterm brain injury and that peripheral inhibition of inflammation with antibiotics, such as vancomycin, reduces the extent of brain injury

Structural basis for a complex I mutation that blocks pathological ROS production

Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury