Mass spectrometry-based proteomics reveals the distinct nature of the skin proteomes of photoaged compared to intrinsically aged skin.

OBJECTIVE: With increasing age, skin is subject to alterations in its organization, which impact on its function as well as having clinical consequences. Proteomics is a useful tool for non-targeted, semi-quantitative simultaneous investigation of high numbers of proteins. In the current study, we utilize proteomics to characterize and contrast age-associated differences in photoexposed and photoprotected skin, with a focus on the epidermis, dermal-epidermal junction and papillary dermis. METHODS: Skin biopsies from buttock (photoprotected) and forearm (photoexposed) of healthy volunteers (aged 18-30 or ≥65 years) were transversely sectioned from the stratum corneum to a depth of 250 µm. Following SDS-PAGE, each sample lane was segmented prior to analysis by liquid chromatography-mass spectrometry/mass spectrometry. Pathway analysis was carried out using Ingenuity IPA. RESULTS: Comparison of skin proteomes at buttock and forearm sites revealed differences in relative protein abundance. Ageing in skin on the photoexposed forearm resulted in 80% of the altered proteins being increased with age, in contrast to the photoprotected buttock where 74% of altered proteins with age were reduced. Functionally, age-altered proteins in the photoexposed forearm were associated with conferring structure, energy and metabolism. In the photoprotected buttock, proteins associated with gene expression, free-radical scavenging, protein synthesis and protein degradation were most frequently altered. CONCLUSION: This study highlights the necessity of not considering photoageing as an accelerated intrinsic ageing, but as a distinct physiological process.

OBJECTIF: Avec l'âge, la peau est sujette à des altérations dans son organisation, et outre le fait d'avoir des conséquences cliniques cela a un impact sur sa fonction. La protéomique est un outil utile pour l'évaluation non ciblée, semi-quantitative, simultanée d'un nombre élevé de protéines. Dans cette étude, nous utilisons la protéomique pour caractériser et comparer les différences associées à l'âge entre une peau photoexposée et une peau photoprotégée, avec une attention particulière sur l'épiderme, la jonction dermo-épidermique et le derme papillaire. MÉTHODES: Des biopsies de peau de la fesse (photoprotégée) et de l'avant-bras (photoexposée) de volontaires sains (âgés de 18 à 30 ans ou de ≥ 65 ans) ont été sectionnées transversalement depuis la couche cornée jusqu'à une profondeur de 250 µm. Suite à une électrophorèse SDS-PAGE, chaque échantillon a été segmenté avant l'analyse par chromatographie en phase liquide couplée à la spectrométrie de masse/spectrométrie de masse. Une analyse des voies de signalisation a été réalisée à l'aide d'Ingenuity IPA. RÉSULTATS: La comparaison des protéomes de la peau des sites des fesses et de l'avant-bras a révélé des différences dans l'abondance relative de protéines. Le vieillissement de la peau de l'avant-bras photoexposée montre une augmentation de 80% des protéines altérées avec l'âge, contrairement à la peau des fesses photoprotégée où une réduction de 74 % des protéines altérées avec l'âge a été mesurée. Sur le plan de la fonction, les protéines altérées par l'âge dans la peau de l'avant-bras photoexposée étaient associées à une structure, une énergie et un métabolisme. Dans la peau des fesses photoprotégée, les protéines associées à l'expression génique, la neutralisation des radicaux-libres, la synthèse des protéines et la dégradation des protéines étaient le plus fréquemment altérés. CONCLUSION: Cette étude souligne la nécessité de ne pas considérer le photovieillissement comme un vieillissement accéléré intrinsèque, mais comme un processus physiologique distinct.

Oxidative stress downstream of mTORC1 but not AKT causes a proliferative defect in cancer cells resistant to PI3K inhibition.

Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells' proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.

Designing disordered materials using DNA-coated colloids of bacteriophage fd and gold.

DNA has emerged as an exciting binding agent for programmable colloidal self-assembly. Its popularity derives from its unique properties: it provides highly specific short-ranged interactions and at the same time it acts as a steric stabilizer against non-specific van der Waals and Coulomb interactions. Because complementary DNA strands are linked only via hydrogen bonds, DNA-mediated binding is thermally reversible: it provides an effective attraction that can be switched off by raising the temperature only by a few degrees. In this article we introduce a new binary system made of DNA-functionalized filamentous fd viruses of ∼880 nm length with an aspect ratio of ∼100, and 50 nm gold nanoparticles (gold NPs) coated with the complementary DNA strands. When quenching mixtures below the melt temperature Tm, at which the attraction is switched on, we observe aggregation. Conversely, above Tm the system melts into a homogenous particulate 'gas'. We present the aggregation behavior of three different gold NP to virus ratios and compare them to a gel made solely of gold NPs. In particular, we have investigated the aggregate structures as a function of cooling rate and determine how they evolve as function of time for given quench depths, employing fluorescence microscopy. Structural information was extracted in the form of an effective structure factor and chord length distributions. Rapid cooling rates lead to open aggregates, while slower controlled cooling rates closer to equilibrium DNA hybridization lead to more fine-stranded gels. Despite the different structures we find that for both cooling rates the quench into the two-phase region leads to initial spinodal decomposition, which becomes arrested. Surprisingly, although the fine-stranded gel is disordered, the overall structure and the corresponding length scale distributions in the system are remarkably reproducible. Such highly porous systems can be developed into new functional materials.

DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions.

We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition.

A HPLC-SRM-MS based method for the detection and quantification of methotrexate in urine at doses used in clinical practice for patients with rheumatological disease: a potential measure of adherence.

Rheumatoid arthritis (RA) is a common autoimmune disease that causes significant disability and reduced life expectancy. The folate antagonist methotrexate (MTX) is first-line therapy for RA when used weekly at low doses (5-25 mg). However, the true rate of adherence to MTX is uncertain. This is in part due to the different methods of measurement of adherence employed with no biochemical test currently available to determine adherence to low dose MTX. Common methods of MTX measurement include immunoassays in patients with high dose therapy, but these assays cross-react with MTX metabolites and lack the sensitivity required to measure adherence to low dose MTX. HPLC-SRM-MS (selected reaction monitoring-mass spectrometry) has several theoretical advantages over immunoassays with improved specificity, minimal cross-reaction and higher sensitivity. The aim of this study was to develop an assay to measure MTX and its major metabolite 7-OH-MTX in urine as a tool to monitor adherence to low dose MTX in clinic. As a proof of concept, urine samples from 4 participants with RA were measured after directly observed therapy. The assay showed improved sensitivity compared to that reported by immunoassays, with low carryover and high within-run precision. In participant samples, MTX was measurable in the urine for up to 105 hours after administration and 7-OH-MTX was detectable up to 98 hours after administration, suggesting that this assay is suitable for the measurement of adherence to therapy. The assay requires minimal sample preparation and can be adopted by other laboratories with minimal study set up.

Renal P2 receptors and hypertension.

The regulation of extracellular fluid volume is a key component of blood pressure homeostasis. Long-term blood pressure is stabilized by the acute pressure natriuresis response by which changes in renal perfusion pressure evoke corresponding changes in renal sodium excretion. A wealth of experimental evidence suggests that a defect in the pressure natriuresis response contributes to the development and maintenance of hypertension. The mechanisms underlying the relationship between renal perfusion pressure and sodium excretion are incompletely understood. Increased blood flow through the vasa recta increases renal interstitial hydrostatic pressure, thereby reducing the driving force for transepithelial sodium reabsorption. Paracrine signalling also contributes to the overall natriuretic response by inhibiting tubular sodium reabsorption in several nephron segments. In this brief review, we discuss the role of purinergic signalling in the renal control of blood pressure. ATP is released from renal tubule and vascular cells in response to increased flow and can activate P2 receptor subtypes expressed in both epithelial and vascular endothelial/smooth muscle cells. In concert, these effects integrate the vascular and tubular responses to increased perfusion pressure and targeting P2 receptors, particularly P2X7, may prove beneficial for treatment of hypertension.

A London experience 1995-2012: demographic, dietary and biochemical characteristics of a large adult cohort of patients with renal stone disease.

BACKGROUND: Kidney stone disease has an estimated prevalence of around 10%. Genetic as well as environmental factors are thought to play an important role in the pathogenesis of renal stones. AIM: The aim of our study was to analyse and report the main characteristics of patients with kidney stones attending a large UK metabolic stone clinic in London between 1995 and 2012. DESIGN: A cross-sectional study. METHODS: Analysis of data from stone formers attending the University College and Royal Free Hospitals' metabolic stone clinic from 1995 to 2012. Demographic, clinical, dietary and biochemical characteristics have been summarized and analysed for men and women separately; trends over time have also been analysed. RESULTS: Of the 2861 patients included in the analysis, 2016 (70%) were men with an average age of 47 years (range 18-87 years) and median duration of disease of 6 years (range 0-60 years). The prevalence of low urine volume, hypercalciuria, hyperoxaluria, hyperuricosuria and hypocitraturia was 5.6%, 38%, 7.9%, 18% and 23%, respectively. The prevalence of several risk factors for stones increased over time. The majority of stones were mixed, with around 90% composed of calcium salts in varying proportion. CONCLUSION: Our findings in a large cohort of patients attending a London-based stone clinic over the past 20 years show differences in distributions of risk factors for stones for men and women, as well as metabolic profiles and stone composition. The impact of most risk factors for stones appeared to change over time.

Drug-induced renal Fanconi syndrome.

A number of therapeutic drugs are toxic to the kidney proximal tubule (PT) and can cause the renal Fanconi syndrome (FS). The most frequently implicated drugs are cisplatin, ifosfamide, tenofovir, sodium valproate and aminoglycoside antibiotics, and the new oral iron chelator deferasirox has also recently been associated with FS. The incidence of full or partial FS is almost certainly under-estimated due to a lack of appropriate systematic studies, variations in definitions of tubular dysfunction and under-reporting of adverse events. The clinical features of FS are amino aciduria, low molecular weight proteinuria, hypophosphataemia, metabolic acidosis and glycosuria. The most serious complications are bone demineralization from urinary phosphate wasting and progressive decline in kidney function. Commonly used tests for kidney function such as estimated glomerular filtration rate and urine albumin/creatinine ratio are not sensitive markers of PT toxicity; patients at risk should thus be monitored with more appropriate tests, and drugs should be stopped or reduced in dose if toxicity occurs. Substantial recovery of PT function can occur after withdrawal of therapy, but this can take months and chronic damage may persist in some cases.

Tropical distal renal tubular acidosis: clinical and epidemiological studies in 78 patients.

BACKGROUND: Distal renal tubular acidosis (dRTA) caused by mutations of the SLC4A1 gene encoding the erythroid and kidney isoforms of anion exchanger 1 (AE1 or band 3) has a high prevalence in some tropical countries, particularly Thailand, Malaysia, the Philippines and Papua New Guinea (PNG). Here the disease is almost invariably recessive and can result from either homozygous or compound heterozygous SLC4A1 mutations. METHODS: We have collected and reviewed our own and published data on tropical dRTA to provide a comprehensive series of clinical and epidemiological studies in 78 patients. RESULTS: Eight responsible SLC4A1 mutations have been described so far, four of them affecting multiple unrelated families. With the exception of the mutation causing South-East Asian ovalocytosis (SAO), none of these mutations has been reported outside the tropics, where dRTA caused by SLC4A1 mutations is much rarer and almost always dominant, resulting from mutations that are quite different from those found in the tropics. SLC4A1 mutations, including those causing dRTA, may cause morphological red cell changes, often with excess haemolysis. In dRTA, these red cell changes are usually clinically recessive and not present in heterozygotes. The high tropical prevalence of dRTA caused by SLC4A1 mutations is currently unexplained. CONCLUSION: A hypothesis suggesting that changes in red cell metabolism caused by these mutations might protect against malaria is put forward to explain the phenomenon, and a possible mechanism for this effect is proposed.

An intact kidney slice model to investigate vasa recta properties and function in situ.

BACKGROUND: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. METHODS: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. RESULTS: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). CONCLUSIONS: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Potential role of serine proteases in modulating renal sodium transport in vivo.

The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.

Alterations of sucrose preference after Roux-en-Y gastric bypass.

Roux-en-Y gastric bypass (gastric bypass) patients reportedly have changes in perception and consumption of sweet-tasting foods. This study aimed to further investigate alterations in sweet food intake in rats and sucrose detection in humans after gastric bypass. Wistar rats were randomized to gastric bypass or sham-operations and preference for sucrose (sweet), sodium chloride (salty), citric acid (sour) and quinine hydrochloride (bitter) was assessed with standard two-bottle intake tests (vs. water). Intestinal T1R2 and T1R3 expression and plasma levels of glucagon-like-peptide 1 (GLP-1) and peptide YY (PYY) were measured. Furthermore, obese patients and normal weight controls were tested for sucrose taste detection thresholds pre- and postoperatively. Visual analogue scales measuring hedonic perception were used to determine the sucrose concentration considered by patients and controls as "just about right" pre- and postoperatively. Gastric bypass reduced the sucrose intake relative to water in rats (p<0.001). Preoperative sucrose exposure reduced this effect. Preference or aversion for compounds representative of other taste qualities in naïve rats remained unaffected. Intestinal T1R2 and T1R3 expression was significantly decreased in the alimentary limb while plasma levels of GLP-1 and PYY were elevated after bypass in rats (p=0.01). Bypass patients showed increased taste sensitivity to low sucrose concentrations compared with controls (p<0.05), but both groups considered the same sucrose concentration as "just about right" postoperatively. In conclusion, gastric bypass reduces sucrose intake relative to water in sucrose-naïve rats, but preoperative sucrose experience attenuates this effect. Changes in sucrose taste detection do not predict hedonic taste ratings of sucrose in bypass patients which remain unchanged. Thus, factors other than the unconditional affective value of the taste may also play a role in determining food preferences after gastric bypass.

Does hypokalaemia cause nephropathy? An observational study of renal function in patients with Bartter or Gitelman syndrome.

BACKGROUND: Hypokalaemic nephropathy has been described in patients with chronic potassium depletion; it is a condition in which proximal tubular vacuolization and interstitial fibrosis occur, resulting in a decline in glomerular filtration rate (GFR) and, in some cases, renal failure. It has been described in patients with chronic diarrhoea, eating disorders, laxative abuse and primary hyperaldosteronism; also occasionally in Bartter syndrome (BS), in which severe hypokalaemia accompanies significant renal sodium and water losses, though rarely in Gitelman syndrome (GS), in which there is equally severe hypokalaemia, but only modest sodium losses. AIM: We hypothesized that hypokalaemic nephropathy may not be due to potassium depletion per se, but persistently elevated circulating levels of aldosterone, possibly with superimposed episodes of renal hypoperfusion. DESIGN AND METHODS: We searched UK and European data sets to retrospectively compare serum and urinary parameters in patients with GS and BS. RESULTS: The patients with GS often had lower serum potassium concentrations than patients with BS, but the BS patients had significantly higher serum creatinine concentrations and lower estimated GFRs (eGFR). BS patients had significantly higher fractional excretions of sodium compared with GS patients, as well as higher plasma renin activities and serum aldosterone levels. CONCLUSION: These findings show that in genetically confirmed cases of BS and GS, the degree of hypokalaemia (as an index of chronic potassium depletion) does not correlate with GFR, and that on-going sodium and water losses, and consequent secondary hyperaldosteronism, may play a more important role in the aetiology of hypokalaemic nephropathy.

Extracellular nucleotides affect pericyte-mediated regulation of rat in situ vasa recta diameter.

AIM: We hypothesized that extracellular nucleotides, established as being released from renal tubular epithelial cells, act at pericytes to regulate vasa recta capillary diameter. METHODS: A rat live kidney slice model and video imaging techniques were used to investigate the effects of extracellular nucleotides on in situ (subsurface) vasa recta diameter at pericyte and non-pericyte sites. In addition, RT-qPCR was used to quantify P2 receptor mRNA expression in isolated vasa recta. RESULTS: Extracellular ATP, UTP, benzylbenzyl ATP (BzATP) or 2-methylthioATP (2meSATP) evoked a significantly greater vasoconstriction of subsurface vasa recta at pericytes than at non-pericyte sites. The rank order of agonist potency was BzATP = 2meSATP > ATP = UTP. The vasoconstriction evoked at pericyte sites by ATP was significantly attenuated by the P2 receptor antagonists suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or Reactive Blue-2 (RB-2). UTP-evoked vasoconstriction at pericytes was attenuated by suramin or RB-2 but not PPADS. Interestingly, suramin or PPADS, when applied in the absence of a P2 receptor agonist, evoked a weak but significant vasoconstriction of vasa recta at pericyte sites, suggesting tonic vasodilation by nucleotides. Significant levels of P2X(1, 3 and 7) and P2Y(4 and 6) receptor mRNA were detected in vasa recta. CONCLUSION: Extracellular nucleotides act at pericytes to cause vasoconstriction of in situ vasa recta. Pharmacological characterization, supported by RT-qPCR data, suggests that P2X(1 and 7) and P2Y(4) receptors mediate nucleotide-evoked vasoconstriction of vasa recta by pericytes. We propose that nucleotides released from renal tubular epithelial cells, in close proximity to vasa recta capillaries, are key in regulating renal medullary blood flow.

Proteomic analysis reveals a novel mechanism induced by the leukemic oncogene Tel/PDGFRß in stem cells: activation of the interferon response pathways.

Objective proteomic analysis offers opportunities for hypothesis generation on molecular events associated with pathogenesis in stem cells. Relative quantification mass spectrometry was employed to identify pathways affected by Tel/PDGFRß, an oncogene associated with myeloproliferative neoplasia (MPN). Its effects on over 1800 proteins were quantified with high confidence. Of those up-regulated by Tel/PDGFRß several were involved in the interferon gamma (IFNγ) response. To validate these observations we employed embryonic and myeloid stem cells models which revealed Tel/PDGFRß-induced STAT1 up-regulation and activation was responsible for modulating the interferon response. A STAT1 target highly up-regulated was ICSBP, a transcriptional regulator of myeloid and eosinophilic differentiation. ICSBP interacts with CBP/p300 and Ets transcription factors, to promote transcription of additional genes, including the Egr family, key regulators of myelopoiesis. These interferon responses were recapitulated using IFNγ stimulation of stem cells. Thus Tel/PDGFRß induces aberrant IFN signaling and downstream targets, which may ultimately impact the hematopoietic transcriptional factor network to bias myelomonocytic differentiation in this MPN.

Albumin uptake in OK cells exposed to rotenone: a model for studying the effects of mitochondrial dysfunction on endocytosis in the proximal tubule?

BACKGROUND: The renal proximal tubule (PT) is clinically vulnerable to mitochondrial dysfunction; sub-lethal injury can lead to the Fanconi syndrome, with elevated urinary excretion of low-molecular-weight proteins. As the mechanism that couples mitochondrial dysfunction to impaired PT low-molecular weight protein uptake is unknown, we investigated the effect of respiratory chain (RC) inhibitors on endocytosis of FITC-albumin in PT-derived OK cells. METHODS: Uptake of FITC-albumin was quantified using confocal microscopy. Cytosolic ATP levels were measured in real time using both luciferin/luciferase assays and measurements of free [Mg(2+)]. Reactive oxygen species production was measured using mitosox. RESULTS: RC blockade produced only a small decrease in cytosolic ATP levels and had minimal effect on FITC-albumin uptake. Inhibition of glycolysis caused a much bigger decrease in both cytosolic ATP levels and FITC-albumin endocytosis. Rotenone led to higher rates of reactive oxygen species production than other RC inhibitors. Rotenone also caused widespread structural damage on electron microscopy, which was mimicked by colchicine and prevented by taxol; consistent with inhibition of microtubule polymerisation as the underlying mechanism. CONCLUSIONS: Endocytosis of FITC-albumin is ATP-dependent in OK cells, but the cells are very glycolytic and therefore represent a poor metabolic model of the PT. Rotenone has toxic extra-mitochondrial structural effects.

Renal function and mitochondrial cytopathy (MC): more questions than answers?

Our knowledge of mitochondrial biology has advanced significantly in the last 10 years. The effects of mitochondrial dysfunction or cytopathy (MC) on the heart and neuromuscular system are well known, and its involvement in the pathophysiology of several common clinical disorders such as diabetes, hyperlipidaemia and hypertension, is just beginning to emerge; however, its contribution to renal disease has received much less attention, and the available literature raises some interesting questions: Why do children with MC commonly present with a renal phenotype that is often quite different from adults? How does a mutation in mitochondrial DNA (mtDNA) lead to disease at the cellular level, and how can a single mtDNA point mutation result in such a variety of renal- and non-renal phenotypes in isolation or combined? Why are some regions of the nephron seemingly more sensitive to mitochondrial dysfunction and damage by mitochondrial toxins? Perhaps most important of all, what can be done to diagnose and treat MC, now and in the future? In this review we summarize our current understanding of the relationship between mitochondrial biology, renal physiology and clinical nephrology, in an attempt to try to answer some of these questions. Although MC is usually considered a rare defect, it is almost certainly under-diagnosed. A greater awareness and understanding of kidney involvement in MC might lead to new treatment strategies for diseases in which mitochondrial dysfunction is secondary to toxic or ischaemic injury, rather than to an underlying genetic mutation.