Control of mitochondrial superoxide production by reverse electron transport at complex I.

The generation of mitochondrial superoxide (O2̇̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2̇̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2̇̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2̇̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2̇̄ production by RET. An analysis of O2̇̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2̇̄ production in both pathological and physiological conditions. We conclude that O2̇̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling

A sensitive method to extract DNA from biological traces present on ammunition for the purpose of genetic profiling

Exploring technological limits is a common practice in forensic DNA research. Reliable genetic profiling based on only a few cells isolated from trace material retrieved from a crime scene is nowadays more and more the rule rather than the exception. On many crime scenes, cartridges, bullets, and casings (jointly abbreviated as CBCs) are regularly found, and even after firing, these potentially carry trace amounts of biological material. Since 2003, the Forensic Laboratory for DNA Research is routinely involved in the forensic investigation of CBCs in the Netherlands. Reliable DNA profiles were frequently obtained from CBCs and used to match suspects, victims, or other crime scene-related DNA traces. In this paper, we describe the sensitive method developed by us to extract DNA from CBCs. Using PCR-based genotyping of autosomal short tandem repeats, we were able to obtain reliable and reproducible DNA profiles in 163 out of 616 criminal cases (26.5%) and in 283 out of 4,085 individual CBC items (6.9%) during the period January 2003–December 2009. We discuss practical aspects of the method and the sometimes unexpected effects of using cell lysis buffer on the subsequent investigation of striation patterns on CBCs

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia

Abstract Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.Peer reviewe

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia

Abstract Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.Peer reviewe

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.publishedVersionPeer reviewe

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease

DNA Methods to Identify Missing Persons

Human identification by DNA analysis in missing person cases typically involves comparison of two categories of sample: a reference sample, which could be obtained from intimate items of the person in question or from family members, and the questioned sample from the unknown person-usually derived from the bones, teeth, or soft tissues of human remains. Exceptions include the analysis of archived tissues, such as those held by hospital pathology departments, and the analysis of samples relating to missing, but living persons. DNA is extracted from the questioned and reference samples and well-characterized regions of the genetic code are amplified from each source using the Polymerase Chain Reaction (PCR), which generates sufficient copies of the target region for visualization and comparison of the genetic sequences obtained from each sample. If the DNA sequences of the questioned and reference samples differ, this is normally sufficient for the questioned DNA to be excluded as having come from the same source. If the sequences are identical, statistical analysis is necessary to determine the probability that the match is a consequence of the questioned sequence coming from the same individual who provided the reference sample or from a randomly occurring individual in the general population. Match probabilities that are currently achievable are frequently greater than 1 in 1 billion, allowing identity to be assigned with considerable confidence in many cases

An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.Peer reviewe