Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria. Economic costs associated with the management of patients with HF is estimated at >US$30 billion annually in the USA alone, and accounts for roughly 2–3% of total healthcare spending globally4. Despite these enormous costs, mortality from HF remains high. Death from HF within 5 years of diagnosis is common despite current optimal medical therapy. Mortality and rehospitalization within 60–90 days after discharge from hospital can be as high as 15% and 35%, respectively5. These event rates have largely not changed over the past 15 years, despite implementation of evidence-based therapy5. HF rehospitalization rates also remain high, with care typically focused on symptomatic relief. Patients with HF are often designated as having either reduced ejection fraction (HFrEF), or preserved ejection fraction (HFpEF). Patients with HFpEF also have poor prognosis after the first diagnosis6. Regardless of the HF aetiology, novel treatments that improve intrinsic cardiac function remain elusive.Heart failure (HF) is associated with substantial clinical burden and economic costs worldwide. The disease is particularly prevalent in elderly individuals, in whom the incidence and associated costs are projected to double over the next 20 years7. Most standard-of-care pharmacological approaches to HF act by reducing workload on the failing heart and, in doing so, attempt to rebalance energy supply and energy demand, albeit to a lower level  and must target the myocardium as the centrepiece of the therapeutic intervention10.Advances in the treatment of ischaemic and valvular heart disease have clearly improved patient survival. The residual cardiac dysfunction and associated comorbidities, however, have led, in the long-term, to the development of HF with attendant poor quality of life. Commonly prescribed HF medications, although beneficial in promoting some symptom relief, often do not fully address the underlying causes of progressive left ventricular dysfunctioner level . Hallmar12. Furthermore, a relative underinvestment in cardiovascular drug development, as well as strategic abandonment by pharmaceutical companies of new therapies for which the risks are perceived to be higher than the rewards, have also contributed to slow development of drugs for HF13. Moreover, the development of effective therapies for HFpEF is imperative to treat this patient population, but the variability in HFpEF phenotypes , and the difficulty in establishing reliable preclinical models of HFpEF, also hinder progress. Despite these obstacles, ample opportunity exists to improve HF treatments, provided the focus is directed towards cardiomyocytes and their intrinsic function.The vast majority of phase III trials in patients with HF conducted in the past decade have been negative, arguably for the same reasons discussed aboveA roundtable meeting was held in Stresa, Italy on 23 October 2015 to discuss the multifaceted problem of insufficient energy production in HF, and the role it has in progressive left ventricular dysfunction. This meeting was attended by academics, clinicians, and representatives from the pharmaceutical industry. The meeting focused on mitochondrial dysfunction as the source of energy deprivation in HF, and how correction of mitochondrial dysfunction using emerging novel therapies might lead to functional improvement of the HF phenotype. This Consensus Statement summarizes the findings from that roundtable discussion.14. Given the astounding energetic cost of cardiac function, this concept is not so far from the truth. Humans produce and consume roughly their body weight in ATP (about 65 kg) every single day15. The heart accounts for only ~0.5% of body weight, but is responsible for roughly 8% of ATP consumption. This high energy flux is dynamic: the heart stores only enough energy to support pumping for a few heart beats, turning over the entire metabolite pool approximately every 10 s even at resting heart rates16. As the most metabolically active organ in the body, the heart possesses the highest content of mitochondria of any tissue. Mitochondria comprise 25–30% of cell volume across mammalian species18, with only the myofilaments being more densely packed within cardiac myocytes. The high mitochondrial content of cardiomyocytes is needed to meet the enormous energy requirement for contraction and relaxation . About 90% of cellular ATP is utilized to support the contraction–relaxation cycle within the myocardium19. ATP-dependent release of actin from myosin is required for both contraction (as myosin heads cycle through cross-bridges with actin) and relaxation. Cellular sequestration of calcium back into the sarcoplasmic reticulum during diastole also requires a tremendous amount of ATP. Cells sustain the energy requirements necessary to support cardiac function through remarkable metabolic supply–demand matching21 , but can also be directly linked to cardiomyocyte injury and death and, therefore, to disease progression. Abnormal mitochondria are a major source of reactive oxygen species (ROS) production, which can induce cellular damage. Abnormal mitochondria can promote programmed cell death through the release of cytochrome 29. Altered mitochondrial biogenesis, fragmentation, and hyperplasia have been observed in studies of human30 and animal models32 of HF. These effects seem to be caused by altered expression of proteins that regulate mitochondrial dynamics33. As many of these factors are ‘master regulators’ of mitochondrial metabolism, these changes might be directly related to the decreased capacity to oxidize fatty acid substrates often seen in HF35.Mitochondria are primarily located within subsarcolemmal, perinuclear, and intrafibrillar regions of the cardiomyocyte. Although they are symbiotic partners with the other cellular compartments, mitochondria are in many ways discrete entities. Mitochondrial dynamics in the form of fission, fusion, and autophagy are highly regulated processes that are essential for energy production and structural integrity of the organelles36. mtDNA encodes 13 protein subunits found within three of the electron transport protein complexes, and a handful of ribosomal and transfer RNAs37. These proteins are made in specialized ribosomes or ‘mitoribosomes’, which are physically attached to the mitochondrial inner membrane38.Mitochondria have their own DNA (mtDNA) and a genetic code that is distinct from the host-cell nuclear DNA. mtDNA is circular in shape, analogous to DNA found in lower organisms, and a primitive fingerprint leftover from bacterial origin. Evolutionary selection pressures have led to mitochondria ‘outsourcing’ almost all their protein-making needs to their cellular hosts. The overwhelming majority (>99%) of mitochondrial proteins come from nuclear-encoded DNA. These proteins are synthesized via cellular protein synthesis machinery, and are actively imported into mitochondria through mitochondrial membrane transporters39. In humans, mitochondria are maternally inherited40, owing to high mitochondrial density in the egg and the active degradation of mitochondria in the sperm during fertilization41. The proximity of mtDNA to sites of mitochondrial ROS generation, poor repair mechanisms, and a lack of protective histones combine to make mtDNA particularly susceptible to oxidative injury and mutation.Many inherited familial cardiomyopathies (both adult and paediatric) are associated with mtDNA mutations37. Mutated mtDNA is found alongside nonmutated copies, leading to mitochondrial ‘heteroplasmy’. The extent of heteroplasmy in mutated mtDNA influences the susceptibility to inherited mitochondrial disease42. Mutated mtDNA can be found in 1 in 200 individuals, a frequency that is 20-fold higher than the incidence of mitochondrial disease. This mismatch indicates that healthy individuals often harbour mutated mtDNA that has no observable phenotypic consequences until a certain mutation threshold is reached37. Although very early in preclinical development, various innovative approaches to reduce the extent of heteroplasmy using genome editing might ultimately lead to effective therapy for HF caused by genetic mitochondrial disease45. Given that mitochondrial abnormalities, such as increased ROS production, altered mitochondrial energetics, and impaired mitochondrial ion homeostasis, are observed in genetic mitochondrial diseases as well as HF, innovative approaches that target mitochondrial dysfunction might share efficacy across these diseases.Mitochondrial genetics contribute to cardiomyopathies by expressing mutant proteins that influence energy homeostasis. With 1,000–10,000 genes per mitochondria (polyploidy), mitochondrial genetics operate on population-based (instead of Mendelian) principlesm)46. Protons that reenter the mitochondrial matrix through complex V  liberate energy that phosphorylates ADP, regenerating ATP. Newly synthesized ATP is rapidly transferred out of mitochondria and energy is subsequently distributed throughout the cell via reversible phosphate exchange networks, primarily catalysed by creatine kinase and adenylate kinase-associated reactions47.The ‘myocardial power grid’ consists of mitochondrial ATP supply that transfers energy throughout the cell along intracellular phosphotransfer buffering systems . MitochoThe evidence that HF involves impaired cellular energy production and transfer is considerable . Among s48. Attempts to improve bioenergetics in HF tend to focus on mitochondrial energy production as a target, because direct augmentation of myocardial creatine with oral creatine supplementation is thwarted by a decreased capacity to transport creatine into the failing cardiomyocytes49. Skeletal muscles also show mitochondrial dysfunction in HF, contributing to the exercise intolerance that characterizes the HF state50. Abnormal mitochondrial function has also been reported in patients with renal insufficiency51, and in patients with insulin resistance52. Given that patients with HF often manifest both renal insufficiency and insulin resistance, treating mitochondrial dysfunction in HF derives benefits that go beyond improving cardiac function 59.Substrate utilization in the failing heart has been extensively reviewed previously61. However, the heart can shift the relative contribution of these substrates in an effort to adapt to varying physiological conditions. Under conditions of low oxygen content, such as ischaemia and HF, ATP content is thought to decrease by as much as 40%3. In HF, fatty acid oxidation and the oxidative capacity of the mitochondria decline, and can no longer maintain sufficient levels of ATP, especially during conditions of increased cardiac workload such as exercise. The failing heart shifts its predominant fuel source from mitochondrial FAO toward glycolytic pathways. This switch is most apparent in late and end-stage HF57, and is 30% more energetically efficient in the failing heart, because more ATP is produced per mole of oxygen during carbohydrate oxidation62. Numerous studies investigating FAO, glucose oxidation, and (to a lesser extent) ketone body oxidation have aimed to establish a metabolic phenotype, underlying molecular mechanisms, and potential therapeutic targets of the failing heart.The heart utilizes different substrates simultaneously to produce energy. Mitochondrial fatty acid oxidation (FAO) is the predominate substrate used in the healthy adult human heart, being responsible for 60–80% of cardiac ATP production, followed by lesser contributions from glucose, lactate, and ketone bodies64. Similarly, tissue from animals and humans with HF has reduced activity of the transcription factor responsible for mitochondrial biogenesis, PPAR-γ co-activator (PGC)-1α65. Because these transcription factors have a critical role in the regulation of cardiac mitochondrial energy production, these data suggest that decreased PPARα and PGC-1α activity might be an important precursor leading to impaired FAO during HF. Therefore, further inhibition of FAO to increase glycolytic flux via PPARα and/or PGC-1α is a plausible therapeutic target. Small-molecule regulators of PGC-1α are needed, and animal models overexpressing the transcription factor are inherently problematic, ostensibly owing to increased mitochondrial biogenesis-induced cardiomyopathy66. Similarly, PPARα antagonists in animal models of HF have yielded inconclusive data67, whereas clinical PPARα ligands are reportedly safe, but their efficacy in a HF population is currently unknown61. Although the safety of PPARα ligands is promising, further evidence demonstrating their efficacy in both animal models and humans with HF is needed.The reduction in fatty acid uptake and FAO that occurs during HF might be owing to dysregulated molecular mechanisms responsible for fatty acid metabolism. For example, the level of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor highly expressed in the heart and responsible for fatty acid transport into the mitochondria and peroxisomes, has been reported to be downregulated in both animal models and humans with HF68. As a result, reducing the availability of circulating free fatty acids via transient adrenergic antagonists might be a viable therapy to inhibit FAO and increase glycolytic ATP production. Traditionally, β-adrenergic receptor antagonists are used in HF owing to their negative ionotropic effects that reduce cardiac workload and spare oxygen by decreasing sympathetic activity68. Many, such as carvedilol, have been clinically shown to lessen infarct size after ischaemia by decreasing sympathetic activity, followed by inhibition of mitochondrial fatty acid uptake and increased glucose oxidation69.Levels of circulating free fatty acids might be higher in the failing heart than under healthy conditions owing to hormonal stimulation. The rise in serum catecholamine levels increases plasma free fatty acid concentrations, and subsequently stimulates FAOO-palmitoyltransferase (CPT) 1, a rate-limiting enzyme in mitochondrial fatty acid uptake68. When intracellular levels of malonyl-CoA are increased, CPT1 is inhibited and mitochondrial fatty acid uptake is stopped70. The intracellular concentration of malonyl-CoA is dependent on the balance between its synthesis via acetyl-CoA carboxylase and degradation via malonyl-CoA decarboxylase. Therefore, the upregulation of acetyl-CoA carboxylase or inhibition of malonyl-CoA decarboxylase would increase intracellular malonyl-CoA levels, and prevent mitochondrial uptake of free fatty acids to reduce FAO. As expected, inhibiting malonyl-CoA decarboxylase in animal models has reportedly improved cardiac function after ischaemia, reduced cardiac FAO, and increased glycolytic flux72. Studies of malonyl-CoA decarboxylase inhibitors in patients with HF are needed.Malonyl-CoA endogenously regulates fatty acid concentrations by controlling the activity of carnitine 58. Compensatory substrate switching towards glucose use has been observed in both animal models and humans59, with a higher contribution coming from glycolysis. Stimulating mitochondrial glucose oxidation, either directly or by inhibiting fatty acid catabolism, has been suggested as a viable therapeutic strategy to compensate for the energetically ‘starved’ failing heart59.Trends in glucose oxidation across the spectrum of HF are more variable, particularly among animal models of HF73. Studies have reported increased ketone utilization in the severely failing heart in humans74. Further research is needed to understand the role of ketone oxidation in the failing myocardium, and to determine whether targeting ketone metabolism is a plausible therapy to improve energetics in HF.Ketone body metabolism also seems to be altered in HF. Ketones are formed in the liver via fatty acid metabolism, and provide a small substrate pool for oxidation within the myocardium. In conditions such as diabetes or starvation, ketone catabolism is upregulated in response to lowered insulin availability and higher fatty acid levels75. Alterations in protein levels modulated by microRNA expression have been proposed to have important implications for glycolysis, β-oxidation, ketone metabolism, the Krebs cycle, and the electron transport chain (ETC)75. For example, increased levels of ROS can alter calcium handling in HF by modifying microRNA that leads to inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2a transcription75. Post-translational modification via lysine acetylation has been suggested to have an important role in metabolic enzyme regulation in the mitochondria59.Novel insights into the regulation of metabolic substrate demand in the heart have been provided through studies of microRNAs and acetylation of mitochondrial lysine residues. Alterations in microRNA levels through any number of upregulation and downregulation events can alter substrate utilization in the heart76. Sustained sympathetic drive and chronically elevated circulating catecholamines — processes that are normally transient to mediate acute increases in cardiac output — are commonly observed in patients with HF (particularly HFrEF)78. Chronic stimulation of β-adrenergic receptors has been directly linked to mitochondrial ROS production through adrenergic receptor-mediated second messenger signalling79,80. ROS-mediated initiation of mitochondria-dependent cell death cascades has been repeatedly observed after chronic sympathetic activation, leading to overall declines in mitochondrial function86. These processes can be amplified by the formation of aminochromes, catecholamine metabolites known to impair mitochondrial redox balance87. Attenuation of HF pathology with β-blockers and rennin–ngiotensin–aldosterone antagonism has resulted in substantial clinical improvements88, and is likely to relieve some of the mitochondrial dysfunction that accompanies increased sympathetic tone. The capacity to complement these existing background therapies with compounds that directly target mitochondrial dysfunction is a potentially promising novel paradigm  toneparadigm .91. ROS are produced at several different sites within cells, both within and outside of mitochondria (reviewed in detail previously95). Mitochondrial ROS production occurs at various sites along the inner mitochondrial membrane as well as in the mitochondrial matrix by components of the ETC and the Krebs cycle, respectively96  can damage proteins and lipids, trigger cell-death cascades, and evoke synchronized collapses in the cellular energy grid97,98. Heightened mitochondrial ROS production and downstream ROS-mediated damage has been reported in patients with HF as well as in preclinical models of the disease101.Cellular ROS production occurs when ROS formation outpaces or exhausts compensatory signals and overwhelms endogenous scavenging systemstively96 . ROS proper se are best characterized by the term ‘hormesis’: small amounts can evoke adaptive signalling and create beneficial, compensatory responses. Modest production of ROS has been shown to mediate beneficial myocardial signalling involved in physiological responses such as (transient) sympathetic drive102, many preconditioning paradigms103, cardiac mitochondrial quality control104, and exercise105. Exercise training is known to augment endogenous ROS-scavenging mechanisms in the heart107, restore bio-energetic efficiency in porcine models of HFpEF108, and improve symptoms and quality of life in trials involving patients with HFrEF110 or HFpEF111. Consistent with the ROS hormesis concept, several studies have noted that administration of high doses of ROS scavengers can abolish the beneficial effects of exercise112,113, including humans taking oral vitamin C or E supplements114.Although ROS are typically associated with pathological states, ROS levels in the heart 115 might be a compensatory mechanism to reduce ROS by ‘uncoupling to survive’116, whereby a reduction in mitochondrial membrane potential is postulated to lower ROS emission from mitochondria. This view is popular and almost dogmatic, but the decrease in ROS production by uncoupling is a prominent effect during mitochondrial state 4 respiration (no ADP). Heart mitochondria, however, are never respiring in state 4. Pathological ROS production in cardio-myocytes is likely to be more closely linked to decreased or collapsed membrane potential and/or depletion of the NADPH pool119, whereby ROS production overwhelms endogenous scavenging through mitochondrial membrane-dependent mechanisms89.Mitochondrial production of ROS depends on the mitochondrial membrane potential. Increased expression of mitochondrial uncoupling proteins in HF121 continues to plague cardiovascular drug development, suggesting that oxygen radical scavenging per se is not a plausible mechanism of action for long-term improvements in HF. Lack of tissue permeability, poor intra-cellular targeting, and ineffective therapeutic doses might contribute to the poor translation of benefits of anti oxidants to date. This approach to therapy, however, might ultimately succeed when novel scavenging compounds that overcome permeability and targeting problems, such as XJB-5–131 , double-blind, placebo- controlled study in patients with HF and showed a reduction in mortality after 2 years of treatment. Although the Q-SYMBIO trial was fairly small, the promising findings triggered interest in the development of other CoQ analogues that more effectively target mitochondria. New quinone conjugates that are tethered to lipophilic, cationic triphenylphosphonium moieties, such as MitoQ, SkQ, and other plastoquinones, might improve the delivery of CoQ to mitochondria143, and have shown some promise in preclinical models of HF144. A potential problem with the use of these compounds is that they are self-limiting, in that they can depolarize mitochondria and inhibit mitochondrial respiration at high concentrations145. Several short-chain synthetic CoQ analogues are also in development, including EPI-743  pool comprises a redox-cycling coenzyme found in the ETC. CoQ is typically synthesized 743 REF.  and ideb4. Cardiolipin decrements are observed in both paediatric150 and adult152 patients with HF. Cardiolipin is essential for the activity of ETC complexes, membrane transporters, mitochondrial ion homeostasis, and ROS production153. Given that most mitochondrial complexes associated with energy production are oligomers composed of many subunits, cardiolipin is proposed to act as molecular ‘glue’ holding these subunits together156. Approaches that target cardiolipin are likely to improve electron transport across the ETC and, in doing so, might be beneficial in treating HF.Aberrant mitochondrial membrane phospholipids in HF are integrally involved in ETC dysfunction. A membrane phospholipid integral to optimal function of the ETC and whose content and composition are altered in HF is cardiolipin. Cardiolipin resides in the inner mitochondrial membrane  and, unl157. MTP-131 has no discernible opioid-receptor activity158, but was found to localize to the inner mitochondrial membrane159, reduce myocardial ischaemia– reperfusion injury161, improve renal function162, and restore skeletal muscle function163. MTP-131 is not a direct ROS scavenger164, and is postulated to act by interacting with cardiolipin165 to interrupt the vicious cycle of ROS-mediated cardiolipin oxidation and subsequent loss of energetics166. MTP-131-mediated improvements in mitochondrial energetics have been observed across a number of different tissues in animal models of disease, including the myocardium164. Of note, MTP-131 can improve mitochondrial bioenergetics by improving respiratory supercomplex formation .A compound that targets cardiolipin in the mitochondria that is currently in clinical development is the cell-permeable peptide MTP-131 . An analogue of MTP-131 (SS-31) was serendipitously discovered by Szeto and Schiller in attempts to identify small peptides with opioid-receptor binding properties167. In complementary studies, MTP-131 administration substantially reduced maladaptive remodelling, preserved cardiac function, lowered β-adrenergic- mediated calcium overload, and restored mitochondrial protein expression170. A substantial improvement in cardiac function with MTP-131 has been demonstrated in a porcine model of HFpEF171 and a canine model of HFrEF172. Beneficial improvements in ejection fraction were associated with improved activity or expression of mitochondrial complexes I, IV, and V, and a normalization of cardiolipin levels172. As the HF syndrome influences many different tissues . Although the opening of the MPTP has historically been thought of as a pathological event leading to cell death, studies now suggest that transient MPTP opening might be a physiological ‘reset’ mechanism to prevent mitochondrial calcium overload. Rare, transient openings of the MPTP have been observed in individual mitochondria of primary cardiomyocytes180. Small, brief MPTP openings were found to be more frequent in HF cardiomyocytes, and were associated with transient mitochondrial depolarization and mitochondrial calcium release. If opening of these pores might be a normal compensatory mechanism akin to ‘pressure release valves’, the concept of treating HF by blocking them becomes increasingly difficult. Ongoing uncertainty regarding the molecular identity of the MPTP further complicates the development of novel therapies that act on the pore185. The MPTP seems to be comprised of ATP synthase (complex V) dimers and to be gated by mitochondrial matrix calcium content via cyclophillin D187.The mitochondrial permeability transition pore (MPTP) is a nonspecific pore that opens in response to increased calcium levels and oxidative challenge, and is associated with ROS production, apoptotic cell death, and mitochondrial dysfunction. Increased proclivity of MPTP opening occurs in both acute and chronic heart disease, and numerous preclinical studies have demonstrated efficacy in cardiac pathology with MPTP blockers, such as cyclosporin, NIM811, and TRO40303  still exist before gene transfer realizes its full translational potential207. Furthermore, increased ROS can oxidize proteins associated with the ryanodine receptor calcium-release channel, which can lead to calcium leaking out of the sarcoplasmic reticulum during diastole208. Increased intracellular sodium levels in HF212 also contribute to poor calcium handling through mechanisms involving sodium–calcium exchange. Given that calcium is central to maintaining bioenergetic supply–demand matching213, sodium overload alters cellular and mitochondrial calcium fluxes and impairs bioenergetic supply– demand matching in HF214. Although very early in development, inhibitors of the mitochondrial sodium– calcium– (lithium) exchanger215, such as CGP-37157, have been shown to improve cardiac function in preclinical models of HF217. Inhibiting the sarcolemmal sodium– calcium exchanger might also be a promising approach, as demonstrated in a preclinical model of HFpEF218.Impaired cellular calcium handling that leads to decrements in excitation–contraction coupling is noted across HF aetiologies, and contributes to poor cardiac mechanics and to arrhythmogenesis219, which prolongs the duration of systole in animal models and in human HF222. Two substantial phase IIb, double-blind, randomized studies comparing omecamtiv mecarbil and placebo have been conducted. In the ATOMIC-HF trial223, omecamtiv mecarbil was administered for 48 h intravenously to patients with acute HF. Overall, the study was neutral (with some evidence of a symptomatic benefit at higher doses), but suggested omecamtiv mecarbil was safe. In the COSMIC-HF trial224, an oral formulation of omecamtiv mecarbil was associated with improvements in cardiac function over 20 weeks, with an effect that persisted for 4 weeks after stopping the drug, suggesting that improved function had produced favourable structural remodelling. Despite the promise of omecamtiv mecarbil, concerns about elevated levels of serum troponin225, metabolic inefficiency226, and impaired cardiac relaxation227 must be assuaged by larger clinical trials to understand fully whether this approach can improve prognosis in HF.Another compound in clinical development to improve cardiac efficiency in HF is omecamtiv mecarbil (CK-1827452). This drug increases the calcium sensitivity of the myofilamentsThe vast majority of HF trials over the past decade have been neutral, and event rates remain unacceptably high. Perhaps most alarming, no proven therapies exist for patients with worsening chronic HF or HFpEF — populations that collectively comprise the majority of the total HF population. Moreover, although systemic blockade of maladaptive neurohormonal responses has improved outcomes in HFrEF, these agents also lower blood pressure and/or heart rate, and development of new haemodynamically active drugs for stepwise addition to existing therapies raises safety and tolerability concerns. Therefore, an ideal novel therapy would be haemodynamically neutral and target the myocardium as the centrepiece of the therapeutic mechanism. In this context, overwhelming evidence from both preclinical and clinical studies indicates bioenergetic insufficiency in HF. Studies using preclinical models of the disease continue to advance our understanding of the cellular and molecular mechanisms that contribute to poor bioenergetics of the failing heart. Considerable potential exists to fill this unmet need, mitigate the economic burdens, and reduce symptoms in patients with HF by focusing on the development of new therapeutic modalities that target mitochondrial abnormalities in HF.


DNA-based snapshots of ancient vegetation have shown that the composition of high-latitude plant communities changed considerably during the late Quaternary. However, parallel changes in biotic interactions remain largely uninvestigated. Here we show how mutualisms involving plants and heterotrophic organisms varied during the last 50,000 years. During 50–25 ka BP, a cool period featuring stadial-interstadial fluctuations, arbuscular mycorrhizal and non-N-fixing plants predominated. During 25-15 ka BP, a cold, dry interval, the representation of ectomycorrhizal, non-mycorrhizal and facultatively mycorrhizal plants increased, while that of N-fixing plants decreased further. From 15 ka BP, which marks the transition to and establishment of the Holocene interglaciation, representation of arbuscular mycorrhizal plants decreased further, while that of ectomycorrhizal, non-mycorrhizal, N-fixing and wind-pollinated plants increased. These changes in the mutualist trait structure of vegetation may reflect responses to historical environmental conditions that are without current analogue, or biogeographic processes, such as spatial decoupling of mutualist partners. Recently, an eDNA metabarcoding data set was used to describe northern high-latitude vegetation during the past 50,000 years. Here, Zobel et al. use the data set to examine how the abundance of key plant mutualistic traits changed during this period and discuss possible environmental drivers. Until now, most attention has been paid to herbivory and specifically its dynamic relationship with vegetation composition.4 Much less is known about the stability, or otherwise, of mutualisms through time. However, the spatial and temporal scales over which such relationships change is an important additional key to understanding ecosystem responses to future climate change5. Palaeodata offer important insights into the long-term trajectory of interactions and the longevity of ecosystems6. Conceivably insights into key plant mutualisms can be gained from examining floristic composition and related plant traits during the climate changes of the late Quaternary.Biotic interactions, including mutualisms, play an important role in determining species’ responses to environmental change and thus mediate community composition and diversity. Through time, both plant life-history traits8. However, many records from north-temperate and boreal regions are dominated by wind-pollinated taxa that are high pollen producers, weighting the signal against insect-pollinated taxa and underestimating species richness in records based on relatively low pollen counts9. Macrofossils can provide a greater level of taxonomic resolution than many pollen records, but they are less common, they provide localized snapshots of palaeovegetation and their preservation varies depending upon the durability of the plant tissue10. Metabarcoding of ancient DNA is an alternative approach to studying past vegetation11 that has the potential to produce taxonomically rich records12. While it has limitations, such as poor resolution of some taxonomic groups13 and representational biases among functional groups14, ancient DNA occurs in most depositional environments, and its analysis can potentially reveal those fractions of plant communities and local floras not reliably retrieved by analysis of pollen or plant macrofossils.Most records of past vegetation derive from fossil pollen, to the extent that records have been collated into comprehensive databases12 generated an extensive environmental DNA (eDNA) data set describing the floristic composition of vegetation at sites across the largely unglaciated northern high latitudes  during the last 50,000 years. They assigned samples to three time periods, representing a compromise that allowed robust statistical comparisons but also reflected key changes in climatic conditions pertinent to the study region, which differed from those of glaciated Eurasia and North America . Because the period 25 to 15 ka BP was comparatively climatically stable (cold and arid) in the study region, Willerslev et al.12 designated it the last glacial maximum (LGM). The pre-LGM period (>25 ka BP) was characterized by fluctuating stadial-interstadial conditions17, while after 15 ka BP (post-LGM), a major climatic transition affected the region, bringing moister and warmer conditions that were a precursor to the establishment of the current interglacial period . Willerslev et al.’s12 results indicated the existence of taxonomically rich pre-LGM vegetation that was dominated by forbs and encompassed taxa that typify both tundra and arctic steppe environments. Taxonomic diversity declined during the LGM, and the LGM flora was largely a subset of the pre-LGM flora. Post-LGM diversity increased, without reaching the pre-LGM level, and comprised fewer forbs and more graminoids and trees than in earlier periods, reflecting a shift from dry tundra-steppe to moist tundra and forest. Willerslev et al.12 linked vegetation structure and composition with megafaunal diet but did not consider other biotic interactions, such as mutualisms. Here, we investigate the representation of mutualisms associated with late-Quaternary vegetation, focusing on three types of mutualistic interaction between plants and heterotrophic organisms: mycorrhizal associations, symbiotic nitrogen fixation, and pollination.Recently, Willerslev et al.20.Mycorrhizae are an ancient symbiosis between soil fungi and plant roots. Several types of mycorrhizal symbiosis are recognized, of which three are widespread. Arbuscular mycorrhizae (AM) are the most ancient type and also the commonest, with > 80% of vascular plant species capable of entering into the symbiosis. Ectomycorrhizae (ECM) and ericoid mycorrhizae (ERM) have evolved multiple times, all more recently than AM, and are only formed by approximately 2 and 1% of vascular plant species, respectively. Non-mycorrhizal status (NM), which is also evolutionarily recent, can be attributed to about 6% of plant species21 and migration23 of AM and ECM trees during the Holocene.With increasing latitude or altitude, predominantly AM vegetation  is replaced by ECM forests with AM understory, then by ECM forests with ERM understory, and finally by ERM dominated heathlands and tundra heaths. One might expect that temporal change in the predominance of mycorrhizal types should follow a broadly analogous pattern to those observed in relation to latitude and altitude, e.g. such that the predominance of mycorrhizal types in historically cooler periods most closely resembles the pattern in contemporary high-latitude vegetation. However, actual data about the occurrence during the late Quaternary of plants with particular mycorrhizal characteristics are scarce and concern differential extinction24. In Europe, at the regional scale, OM plant species are known to prefer drier and warmer habitats with higher soil pH, while FM species have broader distributions26. The benefit of mycorrhizal association may change in relation to CO2 concentration27, but there is no empirical information about mycorrhizal function in relation to historically lower CO2 concentrations or under conditions of carbon starvation. Nor is there information on how the shares of NM, OM and FM plant species have changed through the late Quaternary.If a plant species is involved in mycorrhizal symbiosis, its mycorrhizal status—whether it is obligately mycorrhizal (OM) or facultatively mycorrhizal (FM)—provides an indication of its reliance on the symbiosis2 from the air with the help of i) bacteria collectively referred to as rhizobia, ii) the actinobacteria genus Frankia or iii) cyanobacteria29. In certain conditions, plants hosting symbiotic nitrogen-fixing microbes are major ecosystem drivers31.The local abundance of the N-fixing symbiosis depends on temperature, soil resources and other biotic interactions29, while, at large scales, the rate of biological N-fixation depends on climatic parameters and CO2 concentration29. In current vegetation the share of N-fixing plants in communities tends to decline with increasing latitude32. However, studies describing variations through geological time in the relative abundance of N-fixing plants are scarce (but see31), and we are unaware of any studies relating to the late Quaternary.Symbiotic nitrogen fixation reflects the ability of some plant species to fix gaseous N33. Empirical information about the geographic distribution of pollination modes remains limited. Some studies have shown that the proportion of particular pollination modes in plant communities may depend on altitude and average wind speed35. Dalsgaard et al.36 modelled the impact of historical climate change on current plant-pollinator networks, but we are unaware of similar studies using empirical palaeoecological data. Indeed, it has generally been difficult to assess the relative abundance of different pollination modes in palaeovegetation because wind-pollinated species predominate in pollen records. Using DNA-based techniques to estimate the proportions of wind-pollinated plants in late Quaternary vegetation could provide an important insight into potential biases affecting pollen-based analyses.Alongside predominantly trophic interactions, such as mycorrhiza and biological N-fixation, plant-pollinator relationships represent an important class of mutualistic interaction. The proportion of animal-pollinated species among Angiosperms varies from 78% in the temperate zone to 94% in the tropics12 data set to investigate how the abundance of plants associated with different symbioses changed during the late Quaternary. Contemporary correlates of mutualist trait abundance provide some basis for making general predictions about temporal patterns during the late Quaternary. Since most traits have been shown to vary in relation to latitude we hypothesized that trends in the relative abundance of mutualist types through warmer and cooler periods of the late Quaternary might broadly reflect patterns seen along contemporary latitudinal gradients. However, we note that modern analogues for past vegetation  are incomplete, reflecting the large difference in climatic conditions between glacial and interglacial periods38. Indeed, our analyses show that changes in the relative abundance of mycorrhizal types and statuses, N-fixing plants and insect-pollinated plants in late-Quaternary vegetation do not consistently reflect patterns seen along contemporary latitudinal gradients. Instead, alternative patterns of mutualistic relationship may have been driven by the direct or interactive effects of non-analogous environmental conditions or by biogeographic processes accompanying climate changes.Here, we integrate available data on plant mutualistic traits with Willerslev et al.’sAmong the molecular operational taxonomic units (MOTUs) retained for analysis, 86 (66%) were forbs, 28 (21%) were graminoids, 11 (8%) were dwarf shrubs and 6 (5%) were trees or shrubs; 86 (66%) were AM, 7 (5%) were ECM, 10 (8%) were ERM and 28 (21%) were NM; 58 (44%) were FM and 45 (34%) were OM; 9 (7%) formed N-fixing symbioses and 122 (93%) did not form N-fixing symbioses; 87 (66%) were insect-pollinated and 44 (34%) were wind-pollinated.19, and our data confirmed these correlations . The post-LGM was characterized by fewer forb reads and more reads corresponding to other growth form categories, compared with both of the earlier periods. The proportions of taxa falling into the different growth form categories followed a similar pattern . The average OMI of individual MOTUs was significantly greater than expected at random (P < 0.01), supporting the positioning of MOTUs on the ordination plot —to approximate the position of MOTUs in a climatic niche space defined by the pre-LGM, LGM and post-LGM periods. Niche positioning is calculated as the distance between the mean environmental conditions used by the taxon (its occurrence in different periods) and the mean conditions of the study as a whole  but not along the second (pre-LGM/post-LGM vs LGM) Fig. . The aveThe estimated positioning of different mutualist trait categories in climatic niche space was relatively similar in non-phylogenetic models and in models where phylogenetic signal was co-estimated with other model parameters  and non-mycorrhizal (NM) taxa increased, as did the share of FM taxa. The proportion of N-fixing taxa decreased. Plant species richness increased again in the post-LGM, though it did not reach the level of the pre-LGM12. Notably, there was a further decrease in the share of AM taxa and an increase of ECM and NM taxa. The share of N-fixing taxa increased to approximately pre-LGM levels, though there was no association between the N-fixing trait and MOTU climatic niche positioning. Temporal variations in the pollination trait also received inconsistent statistical support: temporal patterns in the share of pollination types were weak, but wind-pollinated MOTUs were significantly associated with the post-LGM. These results indicate weak support for some contemporary environmental correlates as drivers of mutualist change through the late Quaternary, but no support for others. Our hypothesis that trends in the abundance of mutualisms during cool and warm periods of the late Quaternary would mirror patterns seen along contemporary latitudinal gradients was generally not supported.The distribution of mutualist traits in northern high-latitude vegetation has fluctuated during the last 50,000 years. The relatively taxon-rich vegetation of the pre-LGM was dominated by taxa that form AM, with fairly equal abundances of OM and FM taxa. Most taxa were non-N-fixing during this period. During the LGM, plant species richness decreased40, data-model comparisons41 and ecosystem function reconstructions taking into account the extinction of megafauna4 attest that conditions in the study region for much of the late Quaternary do not have direct analogues today42. Below we consider how such non-analogous aspects of the climate and environment may have contributed to the results we observed. We also consider whether biogeographic processes and biotic interactions within the wider ecosystem might have played a role. While we cannot explicitly address the mechanisms underlying the observed patterns on the basis of descriptive data, we propose what we believe to be plausible explanations.Both physical data43. Trends in precipitation may be an important driver of these changes. High soil moisture and low temperature are associated with NM status and low representation of AM species in contemporary northern-hemisphere vegetation44. Increasing precipitation across the study region from ca. 14 ka BP onwards46 resulted in higher soil moisture, making the post-LGM the wettest of the three periods. This shift is therefore consistent with the observed NM increase and AM decrease in the post-LGM. The changing level of atmospheric CO2 may also have played a role, given the strong increase in CO2 concentrations from as low as 190 ppm during the LGM to ambient post-industrial levels of 300–400 ppm47. The efficiency of AM associations has been shown to decrease with increasing CO2 (34), while ECM plants exhibit a strong positive response to elevated CO227.Trends in the abundance of mycorrhizal types across warmer and colder periods of the late Quaternary did not mirror patterns seen along contemporary latitudinal and elevational gradients. We observed a shift from AM to ECM and NM in the post-LGM, but, interestingly, not to ERM, even though Ericales are most dominant in post-LGM pollen records50. In general, AM particularly enhances plant P nutrition, while ECM allows plants to obtain N19. On this basis, the observed relative abundances of these mycorrhizal types suggest that P-limitation in pre-LGM vegetation may have been replaced by N-limitation in post-LGM vegetation. Increasing precipitation with the onset of interglacial conditions led to the development of more organic soils and to paludification in some parts of the study area51. These changes are likely to have offset, at least in part, increases in mineralization of both N and P with rising temperatures52, although the effect may have been dependent on vegetation type53. Aerts et al.49 showed experimentally that moderate changes in summer temperatures stimulate litter P release while having limited effects on litter N release in sub-arctic ecosystems. As a result, N-limitation of plant growth in such ecosystems may be promoted by warming. While no direct evidence of a transition from P-limitation to N-limitation during the last 50 ka exists, a global comparison of sedimentary nitrogen isotopes and carbon accumulation across the glacial–deglacial transition led McLauchlan et al.54 to conclude that terrestrial nitrogen availability generally declined as global climates warmed at the onset of interglacial conditions.Plant associations with mycorrhizal fungi and N-fixing microorganisms may also have been influenced by variation in the availability of potentially limiting nutrients. Nitrogen is thought to be the most limiting nutrient for plant productivity in many contemporary northern ecosystems, although co-limitation by P is not uncommon16, and considering that CO2 levels and soil C:N ratios were lowest in the LGM54, the share of N-fixing plants might be expected to be moderate during the pre-LGM, low during the LGM and highest during the post-LGM. The recorded fluctuation in the share of N-fixing plant taxa was partially consistent with this because the total share of N-fixing plants was slightly higher during the post-LGM.Because the magnitude of biological nitrogen fixation is positively related to ecosystem evapotranspiration55, so it might be expected that warmer climatic periods have also been associated with higher levels of insect pollination. However, the contemporary latitudinal gradient incorporates a larger temperature range than the temporal gradient of the late Quaternary. Indeed, we found that the proportion of insect-pollinated plants did not vary between climatic periods, and ordination analysis suggested that, if anything, wind pollination was more associated with the post-LGM. However, pollination traits were not strongly associated with plant climatic niche positioning once growth form differences were accounted for. Analogous results were recorded by Moeller et al.56 in relation to a contemporary latitudinal pattern. Indeed, many ectomycorrhizal trees and non-mycorrhizal sedges, which increased during the post-LGM, are wind pollinated. The tendency for wind pollination to increase in the post-LGM may indicate that pollen-based analyses capture a larger fraction of existing diversity in this period compared with earlier periods.Warmer regions of the globe tend to harbour higher proportions of insect-pollinated plants37 and the extinction of certain megaherbivores4. The onset of the post-LGM represents a period when temperate organisms recolonized higher latitudes and boreal floras occupied many areas previously dominated by arctic floras. This spatial reorganization was reflected in high temporal rates of compositional change in plant communities in eastern North America57 and Europe58. The post-LGM vegetation communities investigated here also exhibited high spatial turnover and differed from those that had been locally present during either the pre-LGM or the LGM12. Among the biological communities that (re)colonized local sites as the climate warmed during the post-LGM, some potential mutualistic partnerships may not have re-formed if the taxa involved possessed differing dispersal abilities. Analogous disconnects between potential partners can occur during primary succession when plants and AM fungi colonize new sites60, but it is unclear to what extent such processes occur over much larger temporal and spatial scales23. Nonetheless, the high proportions of NM and FM taxa in the post-LGM are consistent with this scenario.Besides important changes in the abiotic environment, the late Quaternary has been characterized by dynamic patterns of organism distribution, including individualistic species responses to climate change42. Changes in the mutualist trait structure of vegetation during the post-LGM—such as the increase of ECM woody plants and decrease of predominantly AM forbs—may have been to some extent related to a reduction in herbivory and disturbance. Nonetheless, it is notable that mycorrhizal status remained a significant predictor of species responses to climatic change even after accounting for the effect of growth form.Major vegetation changes marking the transition to interglacial conditions were broadly coincident with the extinction of megaherbivores in northern Eurasia and North America61. If similarly conserved but unmeasured traits were critical to plant survival under late-Quaternary conditions, such traits might also underlie the patterns observed here. However, we note that mycorrhizal traits—specifically mycorrhizal status—were significant predictors of taxon responses to climate change, even when the effects of other mutualist traits and growth form were accounted for. Therefore, we consider the evidence for a direct link between mycorrhizal traits and taxon responses to be the strongest to emerge from these analyses, although responses associated with other traits should not be discounted.Incorporation of phylogeny into models decreased the precision of estimated trait–environment relationships. Where phylogenetic and non-phylogenetic models suggest similar relationships but with stronger effects in the non-phylogenetic model, this may indicate that trait and niche are to some degree phylogenetically conserved, but that some differentiation is apparent within recently emerged clades12, which, allied with the moderate total sample size, meant that we did not have high power to detect short-term or regional variations. We therefore do not dismiss the possibility that mutualisms across different regional biotas within the study area responded differently to the environmental changes of the last 50,000 years.Our results make the implicit assumption of scale-independent correlation between environment and traits, i.e., a consistent relationship throughout the study region. To the degree that we were able to assess this, the data were largely consistent with this assumption. However, it should be acknowledged that the available sample set provided spatially and temporally uneven coverage62 that incorporate information about current relationships between organisms and the abiotic environment, usually climate parameters. The results of this study show that temporal changes in the abiotic environment were paralleled not only by shifts in floristic composition but by changes in the structure of mutualist relationships among late-Quaternary plant communities. In general the patterns of mutualist trait abundance seen along contemporary latitudinal gradients did not make good predictors of variations in trait abundance between the cooler and warmer periods of the late Quaternary. We have attempted to provide plausible ad hoc explanations for the observed patterns. The complexity of observed responses, even based on a relatively small suite of traits, indicates how features not directly related to climate may be important in predicting trait distributions, and hence floristic composition, in communities.Many forecasts of the impact of climate change on biodiversity are made using macroecological or species distribution models12, who identified plant species or species groups  from 242 permafrost sediment samples collected from 21 sites in the northern high latitudes. Of these, 221 samples came from northern Siberia, Alaska and northwest Canada, regions left largely ice-free during the last glacial maximum that preserve older deposits. Sample radiocarbon ages ranged from modern to approximately 50 ka BP63. Some of the oldest sample ages (40–50 ka BP) may actually be infinite, though most samples are stratigraphically consistent with an MIS-3 designation.We used data from Willerslev et al.40, under cold, dry climates. The sampled deposits were incorporated into the permafrost zone after deposition, but each 100-mm diameter sample represents years of accumulation and may have been subject to bioturbation/cryoturbation over a few centuries prior to freezing, depending upon active-layer depth and sediment accumulation rate . Samples were collected away from the contacts between ice wedges and sediment to ensure that material that may have been moved vertically in relation to frost-cracking and ice-wedge growth was not sampled. Although sites are widely separated, from their current landscape position, many localities were sited on low-lying interfluves meaning that the sampled landscape was more homogenous than if all landscape units  were sampled evenly. These landscapes supported a diverse Pleistocene megafauna64. Post-LGM samples were more varied: some were from yedoma, others from lake sediment, peat and soil. This reflects a major rearrangement of northern landscapes and soils, which featured cessation of loess deposition, development of wetlands and organic soils (with a concomitant reduction in nutrient availability), the northward spread of boreal forest into previous treeless vegetation, and the extinction of many megafaunal taxa. While their origins are more varied, the samples nevertheless record the consequences for vegetation composition of this major environmental shift.Samples were assigned to a simplified temporal classification relevant to the climate history of the study region: >25 ka BP, 25 to 15 ka BP, <15 ka BP, and given the labels pre-LGM, LGM and post-LGM for ease of reference. Samples dating to the pre-LGM and LGM were largely derived from yedoma, ice-rich silt deposits that accumulated, mostly as a result of nutrient-rich loess deposition12 sequenced the P6 loop of the chloroplast trnL (UAA) intron, generating 14,601,839 reads after stringent quality filtering. Reads were assigned to MOTUs present in Arctic and boreal trnL taxonomic libraries13 or GenBank on the basis of sequence similarity (using the programme ecotag65). Rare MOTUs (<5 reads) were omitted12. In total a match was found for 7,738,725 reads and these were presented in Supplementary Table 12.Willerslev et al.66, Willerslev et al.12 used the quantitative information in their data as a proxy for MOTU abundance. We use the same time period classification and quantitative data in our analysis.Because the relative number of reads of a given taxon has been shown to be a sufficiently good proxy of its relative cover or biomass in a vegetation sample12 used information in the BiolFlor database67 to assign MOTUs to the following growth form categories: forbs, graminoids , dwarf shrubs or other woody plants (trees and shrubs). Where MOTUs could only be linked with species groups or higher-level taxa, and all constituent species had the same growth form, this was allocated to the MOTU; otherwise the MOTU was left ‘undefined’. Growth form was allocated to 147 of the 154 identified MOTUs. We used the same approach to compile information on MOTUs for mycorrhizal traits, N-fixation and pollination mode traits from available databases  and additional literature searches.Willerslev et al.26 to Akhmetzhanova et al.68, Wang and Qiu69 and Harley and Harley70—as the more recent data sets generally cover, update and correct earlier information. This process was done carefully to avoid errors propagating in the literature. We performed a review of the mycorrhizal trait data set, limiting possible sources of error as indicated by Brundrett20 and quality controlling the data as described in Bueno et al.25 Four and six MOTUs were allocated to the ‘undefined’ category for mycorrhizal status and type, respectively.To assign a category of mycorrhizal type  and status  to each plant taxon (NM was considered both in the context of types and status), we compiled mycorrhizal information from published data sets, starting with the most recent before moving to progressively older sources—from Hempel et al.To identify which MOTU component plant species were involved in N-fixing mutualisms, we used data from the BiolFlor database and an additional literature search. MOTUs were assigned to two categories: involved or not involved in N-fixing mutualism. Four MOTUs were undefined.72 and there is insufficient information available to classify such species separately in our data set. Because those taxa in our data set that are known to exhibit some degree of self-pollination or agamospermy have insect pollination syndromes , we classified them as insect-pollinated. Nine MOTUs were ‘undefined’.We categorized MOTUs according to the recorded pollination mode in the BiolFlor database. We considered only two major pollination modes: (1) wind pollination and (2) insect pollination. Although many arctic plant populations are predominantly self-pollinated, mixed-mating , or partly or fully agamospermous, this may vary within a species across its rangeAfter compiling the trait database, we removed 14 MOTUs (2226214 reads) that could not be defined for all traits and five samples that no longer contained any MOTU data. This left a data table containing 216 samples , 131 MOTUs  and 5026792 reads . The precision of taxonomic and trait composition estimates was similarly high in all periods  and pollination mode; by contrast, we assumed that ERM mycorrhizal type and N-fixing associations, which are phylogenetically conserved, should be reliable. Thus, for each susceptible trait we randomly selected species corresponding to a given error rate (we considered rates between 1 and 20%). For these species, we reassigned the trait category to a randomly selected alternative category. We repeated this process 1000 times at each error rate to generate replicate data sets with a given level of randomly introduced error. We then calculated the proportional abundance of each trait in every sample and correlated these values with the trait abundance observed in the original data. We also recalculated the major analyses described below assuming a 10% error rate in susceptible traits. We found that while trait abundance varied slightly between data sets using original and random trait errors, the trait responses to climatic period remained very similar, with the possible exception of ECM, which was less strongly associated with the post-LGM in data sets incorporating random error  almost certainly include errors resulting from misidentification of traits and incorrect transfer of information among sources. The level of error among mycorrhizal trait categorizations has been estimated at 10% of species73. We used bootstrapping to estimate confidence intervals around the shares of different categories: we resampled the raw data table 74) to test the null hypothesis of no difference in trait category distribution (both number of taxa and number of reads per category) between climatic periods.To assess the abundance of different mutualist traits in different climatic periods , we calculated for each sample the relative share of each mutualist category in the MOTU list, as well as the relative abundance of MOTUs in each mutualist category, estimated on the basis of read frequency. The shares of different categories among the total pool of reads recovered from a sample resembles what is known as the community weighted mean in plant community ecology39). Unlike some other constrained ordination techniques, it is applicable to environmental gradients of any length and is not influenced by variations in site richness. A recent review of methods proposed OMI as a stable and sensitive tool for investigating trait–environment relationships75. We used OMI to generate a constrained ordination of MOTU abundance in response to climatic period . The species scores from this ordination thus potentially represented MOTU locations in a two-dimensional ‘climatic’ niche space, with the first axis representing a gradient from pre-LGM and LGM on one hand to post-LGM on the other; and the second axis representing a gradient from pre-LGM and post-LGM on one hand to LGM on the other. We used a Monte Carlo randomization test to test whether average MOTU OMI was greater than would be expected at random (i.e. whether the climatic gradients had significant explanatory power39).We also used a constrained ordination approach—the outlying mean index (OMI)—to identify the mutualist traits associated with MOTU-level responses to climatic period. OMI approximates niche positions for species  by calculating the distance between the mean conditions used by a species and the mean conditions available in a study , we tested the null hypothesis of no difference in MOTU climatic niche location in relation to mutualist trait category. We constructed models where the effects of single explanatory traits  were (i) tested in isolation, (ii) tested after accounting for growth form ; and (iii) tested after accounting for growth form and non-confounded mutualist traits . To identify the climatic periods associated with particular trait categories, we tested differences between trait categories in the MOTU scores on the separate individual axes. To test the null hypothesis of no difference between categories, we used Dunn’s non-parametric test, since the residuals from parametric models were not normally distributed.12 demonstrated spatial distance decay in the compositional similarity of plant communities in the permafrost data set. Spatial autocorrelation can lead to inflated Type I error in statistical hypothesis tests, while spatial dependence in trait–environment relationships limits the generality of global estimates of trait–environment correlation77. We assessed the degree of spatial autocorrelation and scale-dependence in trait–climate relationships using multi-scale ordination (MSO)77 and Mantel correlograms. Multi-scale ordination is a method based on canonical correspondence analysis that decomposes total, explained and residual variance at different spatial scales. A systematic difference between total variance and the sum of residual and explained variance can indicate scale-dependent species-environment correlation. To construct correlograms, we used distance-based redundancy analysis (Bray-Curtis distance), which is similar to PERMANOVA but allows the calculation of a residual matrix for use in the correlogram. The MSO procedure also generates an estimate of the degree to which residual variance is underestimated as a result of autocorrelation. We used these values to estimate the influence of measured autocorrelation on Type I error rates in PERMANOVA.Willerslev et al.61. We constructed a phylogeny to represent approximate relationships between the plant MOTUs used in this analysis. To do this, we used a backbone supertree of the European flora78. We first identified MOTUs that corresponded to single plant species that were present in the backbone phylogeny (n = 26). We then grafted terminal branches onto the tree to represent MOTUs that comprised multiple species or higher taxa. For each such MOTU we attached the terminal branch to a node in the phylogenetic tree representing the lowest taxonomic level among genus (75 MOTUs), tribe (7 MOTUs), subfamily (5 MOTUs) and family (18 MOTUs) that comprised all component taxa of the MOTU and was identifiable in the tree. We then pruned all redundant species in the tree to leave a phylogeny containing 131 MOTU tips (Supplementary Fig. 79.Analytical approaches that incorporate phylogenetic relationships between taxa can help to link the evolution of functional traits with adaptation to abiotic or biotic environments. Such ‘phylogenetic correction’ can provide a surrogate of functional trait differences between taxa, and may be particularly important in the presence of missing or complex trait information12. Additional data are available from the corresponding author upon request.The data set of this study is published by Willerslev et al.Supplementary InformationPeer Review File


The occurrence and features of skin impressions in a sauropod footprint, the largest (>50 cm in diameter) reported to date for this taxon, from the Lower Cretaceous Haman Formation (Albian) in Korea are described, and its preservation and paleoenvironmental implications are interpreted. The skin impression-bearing deposits are floodplain sediments formed by sheetflood processes. The large impression is preserved in silty mudstone with microbial lenses and wisps overlying a planar- to cross-laminated and fine-grained sandstone to siltstone bed. The paleoenvironment of the skin impression-bearing deposits is interpreted as a saline sandflat to mudflat where microbial mats can form around lakes or ponds under semi-arid paleoclimatic conditions with alternating wetting and drying intervals. These paleoenvironmental conditions would have permitted the distinct preservation of skin impressions in a dinosaur footprint. The observations here suggest that some sauropod dinosaurs in the Cretaceous had a well-developed polygonal skin texture covering nearly the whole of their foot pads, as seen in modern elephants, which would increase stability when walking on muddy and wet ground. Skin impressions in dinosaur footprints have been documented from only a few isolated casts of footprints11 and tracks13. The Cretaceous fluvio-lacustrine deposits in Korea contain innumerable dinosaur tracks14. However, skin impressions in dinosaur footprints have very rarely been observed in these tracks16. Although several casts of dinosaur footprints are preserved in the Cretaceous deposits of China18, few skin impressions have been observed. Here, we report a large and distinctly preserved skin impression on the cast of a foot pad print of a sauropod dinosaur. We also describe some diffuse and partial skin impressions in other dinosaur footprints. These findings provide insight into the unique conditions for the preservation of skin impressions in dinosaur footprints and the paleoenvironmental and paleoecological implications of these insights.Although several distinct skin impressions in Mesozoic reptile footprints have been documented11. However, the common presence of distinctly preserved dinosaur footprints without skin impressions and the very limited number of impressions on numerous casts of dinosaur footprints suggest that specific preservation conditions may have limited the occurrence of footprints with skin impressions.Dinosaurs had foot pads, but it is unclear why skin impressions in dinosaur footprints are extremely rare. Some studies have suggested that the predominance of dinosaur tracks as underprints or overtracks, the inadequate preservation conditions, or the erosion might explain the rare occurrence of these skin impressionsHere, an unusually large (>50 cm in diameter) distinct skin impression in a sauropod footprint is documented in Early Cretaceous floodplain deposits (Haman Formation) exposed in Gunbook-myeon, Haman-gun, Gyeongsangnam-do, Korea Figs and 2. A The skin impression-bearing sediments are floodplain deposits formed by sheetflood processes. The deposits consist of interlaminated to interbedded fine-grained sandstone to siltstone and mudstone, planar to cross-laminated fine-grained sandstone, and calcareous silty mudstone Fig. . CurrentIn the purple-coloured planar to cross-laminated fine-grained sandstone beds with mudstone films, a large skin impression and its cast, with a diameter of approximately 50 cm and a distinct polygonal texture, are preserved in a subcircular and very shallow (approximately 1 cm deep) footprint of a sauropod dinosaur Fig. . The speThe cast of the skin impression is composed of raised hexagonal polygons ranging from 6 mm to 18 mm in width. In general, the sizes of the polygons decrease radially. The polygonal profile has a distinct V-shape with a depth < 1 mm . The raised hexagonal surfaces are generally arranged in rosettes, where any one polygon is in contact with five to seven other polygons. In general, the distinctiveness decreases as the impression-bearing surface becomes flatter.A polygonal skin impression with rather diffuse edges is also preserved in another subcircular footprint with a very shallow depression Fig. . PartialThe partial preservation of polygonal skin impressions was also observed in another shallow footprint in the interlaminated fine-grained sandstone to siltstone and mudstone bed  installed at PNU Core Research Institute was used for SEM analyses of the skin impression-bearing layer. All the samples were Pt-coated for SEM analysis. The operating conditions were 10 keV for SEM imaging and 15 keV for 30 live seconds for the EDS analyses. All the specimens are stored at the Department of Earth and Environmental Sciences of Pukyong National University, Busan, Republic of Korea.In the field, the skin impression-bearing deposits were measured and photographed. The rock samples for sedimentological and taphonomic analysis were cut with a rock saw and polished. Thin sections were also made and were studied using an optical microscope (Nikon Optiphot2-POL). Photomicrographs were made with a Nikon Coolpix P50 for the thin sections and a Nikon Coolpix P300 and Samsung NX300 for the rocks. A ZEISS SUPRA™25  equipped with an Oxford X-max 80-mmSupplementary information


Vitellaria paradoxa (Gaertn C. F.), or shea tree, remains one of the most valuable trees for farmers in the Atacora district of northern Benin, where rural communities depend on shea products for both food and income. To optimize productivity and management of shea agroforestry systems, or "parklands," accurate and up-to-date data are needed. For this purpose, we monitored120 fruiting shea trees for two years under three land-use scenarios and different soil groups in Atacora, coupled with a farm household survey to elicit information on decision making and management practices. To examine the local pattern of shea tree productivity and relationships between morphological factors and yields, we used a randomized branch sampling method and applied a regression analysis to build a shea yield model based on dendrometric, soil and land-use variables. We also compared potential shea yields based on farm household socio-economic characteristics and management practices derived from the survey data. Soil and land-use variables were the most important determinants of shea fruit yield. In terms of land use, shea trees growing on farmland plots exhibited the highest yields  while trees growing on Lixisols performed better than those of the other soil group. Contrary to our expectations, dendrometric parameters had weak relationships with fruit yield regardless of land-use and soil group. There is an inter-annual variability in fruit yield in both soil groups and land-use type. In addition to observed inter-annual yield variability, there was a high degree of variability in production among individual shea trees. Furthermore, household socioeconomic characteristics such as road accessibility, landholding size, and gross annual income influence shea fruit yield. The use of fallow areas is an important land management practice in the study area that influences both conservation and shea yield. Vitellaria paradoxa (Gaertn C. F.), commonly known as shea tree produces an edible fruit that is the source of one of Africa’s most ancient food oils. Shea trees are indigenous to semi-arid and sub-humid savannas of sub-Saharan Africa (SSA), occurring on nearly 1 million km2among 18 African countries50] traceV. paradoxa is a slow growing species, requiring 15 to 20 years before initial flowering [Anacardiumoccidentale L., Eucalyptus spp. and Tectonagrandis L.f. for plantation projects[The potential benefit from shea parklands in terms of primary production and smallholder farmers livelihoods depend on management. Therefolowering , 61 and lowering . The dellowering , 17, 62  projects.Vitellaria paradoxa parklands are one of the dominant features of the Sudan savannah. The species plays a very important role in the economic and social life of smallholder farmers in SSA. Currently, there is a renewed interest in protecting and improving shea resources as domestic and international demand for shea kernel and shea butter increases. This attempt to characterize shea tree fruit yield in Atacora District was successful in the sense that observed fruit yields were comparable to records from other shea producing areas. Findings in northern Benin provide knowledge on the considerable potential for yield improvement through adequate management practices. Soil groups and management practices should be considered for optimizing shea trees yields, whereas certain household socio-economic characteristics  were found to influence farmers’ decisions regarding management practice choices. Our findings emphasize the cyclical nature of shea productivity, but the long-term patterns continue to be poorly understood. The erratic nature that characterizes fruit yield implies that there is room for improvement provided adequate management is applied. Interestingly, land-use and soil group coupled with dendrometric parameters and household characteristics governing management strategies can be used to predict the total yield of shea parklands. Long-term research efforts of the described phenomenon are needed to determine the patterns of shea productivity and to improve the potential for using physical and morphological parameters for predicting shea yield. The reluctance among farmers to plant shea trees, owned not only to its lengthy juvenile phase, but also to taboos against planting shea trees and to the difficulty in growing them due to the recalcitrant seed. This emphasizes the need for dissemination of improved materials and adequate management practices.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Owing to their two-dimensional confinements, silicon nanowires display remarkable optical, magnetic, and electronic properties. Of special interest has been the development of advanced biosensing approaches based on the field effect associated with silicon nanowires (SiNWs). Recent advancements in top-down fabrication technologies have paved the way to large scale production of high density and quality arrays of SiNW field effect transistor (FETs), a critical step towards their integration in real-life biosensing applications. A key requirement toward the fulfilment of SiNW FETs’ promises in the bioanalytical field is their efficient integration within functional devices. Aiming to provide a comprehensive roadmap for the development of SiNW FET based sensing platforms, we critically review and discuss the key design and fabrication aspects relevant to their development and integration within complementary metal-oxide-semiconductor (CMOS) technology. Howevmeasured a.A completed SiNW FET biosensor device is a complex sensing system, where each of the functional components  and detection method are inter-connected and influence each other b. TherefHigh quality SiNW FETs with various sizes, impurities densities and crystal orientations can be prepared in large quantity using chemical synthesis, for example vapor–liquid–solid synthesis route ,27,28. H10 wires/cm2) −15) usin ketones ,179.Airborne biological transfection is critical not only for public health but also for security concern. Real-time monitoring of airborne biological contaminants using SiNW FET incorporated in wireless modules was also reported . Aerosol2+, Cd2+, Pb2+, and Cu2+) is critical in water monitoring, food processing as well as medicine. By functionalizing SiNW FET with 3-mercaptopropyltriethoxysilane (MPTES), Lou et al. demonstrated the highly sensitive detection of Hg2+ and Cd2+ in aqueous solution at concentrations of 10−7 and 10−4 M, respectively [2+ and Cu2+ could also be achieved using metal an ion selective oligonucleotide modified SiNWs-FETs device [2+ and 10 nM for Cu2+ were obtained. Although the specificity of such sensor was not specified, this study illustrated the promising nature of SiNWs-FET sensing platforms for water pollution monitoring and clinical toxicology applications.Due to their extreme toxicity, the detection of contaminant metal ions , a spin-off company from Harvard University (US), first introduced SiNW FET biosensor prototypes for various application in cancer diagnostic, virus and biological toxin detection. The company provides a complete solution from sample processing, on-chip platform, readout instrument as well as data processing . As presDespite significant technological advances, the commercialization of CMOS compatible SiNW FET biosensor technology is still in the early phase. Similar to other solid-state sensing technologies, significant challenges remain to be solved and further improvements are required to fulfill the intrinsic promises of SiNW FET sensing platforms and allow for their routine implementation. Thanks to the progress in microfabrication technology, large-scale patterning and CMOS integration of SiNW FET is no longer an issue. However, practical challenges associated with high manufacturing cost still need to be resolved. In addition, quality assurance in large scale fabrication of such device is also required to insure the required device homogeneity necessary for regulatory accreditation. Another significant challenge is the measurement paradigm. In the conventional DC measurement in SiNW FET platform, the total conductance changes are measured against charged target molecules concentration in low ionic solution. Development of either new sample processing or new measurement approaches are needed toward fast, reliable and reproducible diagnostic devices. High performance multiplexed conductivity measurement of SiNW FET biosensor also remains an issue in real-life implementation, and the development of bench-top multiple channel readout with ultra-low-noise and high resolution is required.The development of SiNW FET molecular biosensing platforms has been critically discussed from their design and fabrication to functionalization perspectives. Fabrication of high performance SiNW FET biosensor requires the fine balancing of many key factors that influence their sensitivity, stability and reliability. It is therefore difficult to define the ideal characteristics of SiNW FETs and rather specific designs should be considered depending on the targeted applications. As a general rule, SiNW devices with low numbers of nanowires, low doping concentrations and short nanowire channel lengths are expected to display maximal sensitivity but may be limited by lack of stability. Increasing the SiNW surface to volume ratio is also expected to improve the device sensitivity but the effect of the cross-sectional dimension on reproducibility and reliability remains unclear. Further theoretical and experimental studies are required to fully elucidate the structure–function relationship of SiNW FET sensors. Choosing optimum fabrication route, packaging strategy, sample delivery and readout methods requires a careful balancing between benefits and drawbacks of each approaches. These technological keynotes must be taken into account in view of the specific applications. Despite several challenges that still need to be addressed, SiNW biosensing platforms are rapidly being developed in the private sectors, yielding the prospect of an imminent paradigm shift in medical diagnostic and environmental monitoring.


Maternal influenza immunization has gained traction as a strategy to diminish maternal and neonatal mortality. However, efforts to vaccinate pregnant women against influenza in developing countries will require substantial investment. We present cost-effectiveness estimates of maternal influenza immunization based on clinical trial data from Bamako, Mali.We parameterized a decision-tree model using prospectively collected trial data on influenza incidence, vaccine efficacy, and direct and indirect influenza-related healthcare expenditures. Since clinical trial participants likely had better access to care than the general Malian population, we also simulated scenarios with poor access to care, including decreased healthcare resource utilization and worse influenza-related outcomes.Under base-case assumptions, a maternal influenza immunization program in Mali would cost $857 (95% UI: $188-$2358) per disability-adjusted life year (DALY) saved. Adjusting for poor access to care yielded a cost-effectiveness ratio of $486 (95% UI: $105-$1425) per DALY saved. Cost-effectiveness ratios were most sensitive to changes in the cost of a maternal vaccination program and to the proportion of laboratory-confirmed influenza among infants warranting hospitalization. Mean cost-effectiveness estimates fell below Mali’s GDP per capita when the cost per pregnant woman vaccinated was $1.00 or less with no adjustment for access to care or $1.67 for those with poor access to care. Healthcare expenditures for lab-confirmed influenza were not significantly different than the cost of influenza-like illness.Maternal influenza immunization in Mali would be cost-effective in most settings if vaccine can be obtained, managed, and administered for ≤$1.00 per pregnant woman. Maternal immunization has emerged as a potential strategy to mitigate maternal and neonatal mortality. In addition to protecting the pregnant mother, maternal vaccination may protect the fetus and infant in the critical first months of life through transfer of IgG antibodies across the placenta . In highThe risk of complications from influenza infection is significantly higher in pregnant women  and infaAdoption of maternal influenza immunization programs in low-income countries will require a firm case for investment. The cost-effectiveness ratio (CER) will depend on the health benefits of vaccination including decreased influenza-related morbidity and mortality for mothers and their infants, the economic benefits of vaccination averting influenza-related healthcare expenditures, and the programmatic costs of vaccination including supplies as well as the infrastructure to manage and administer influenza vaccine to pregnant women. We collected prospective data on direct and indirect costs of laboratory confirmed influenza (LCI) and influenza-like illness (ILI) incurred during the trial in Mali. We combined these results with epidemiological and vaccine efficacy data  to paramWe built a decision tree model of the costs and benefits of maternal influenza immunization. All benefits of maternal influenza vaccine were assumed to stem from prevention of laboratory-confirmed influenza in the pregnant mother, the infant, or the post-partum mother. After an initial decision to either vaccinate or not vaccinate the pregnant mother, further events including influenza infection in the pregnant woman, infant, or post-partum mother proceeded in a probabilistic manner . At eachAll calculations were based on a societal perspective. To reduce the likelihood of overestimating the cost-effectiveness of maternal influenza immunization, we only included DALY losses from premature death  and costAttack rates of influenza in mothers and infants, vaccine efficacy in mothers and infants, the risk of infant hospitalization after contracting LCI, and outpatient and inpatient costs of illness for mothers and infants were all taken from primary data collected during a phase 4 randomized controlled effectiveness trial conducted in Bamako, Mali from September 2011 –January 2014 that determined the efficacy, safety, and immunogenicity of trivalent inactive influenza vaccine (TIV) in pregnant women and their infants up to 6 months of age. Full study details are described elsewhere[rd trimester at 6 community and referral health centers in Bamako, Mali. After randomization, women were vaccinated with either TIV or meningococcal conjugate vaccine (MCV). From enrollment until the infant reached age 6 months, field personnel performed weekly visits, during which the participating woman and infant  had their temperatures measured and were evaluated for ILI. When the ILI case definition was met nasopharyngeal/oropharyngeal swabs and a malaria blood smear were obtained, and a team dedicated to estimating costs of illness was contacted. For participants treated as outpatients, study personnel visited the home every 2–5 days for the duration of the episode to ascertain the direct costs of illness  and indirect costs of illness (defined as the number of workdays lost by each family member multiplied by that family member’s average daily earnings). Additionally, all medications prescribed by study physicians and filled at the recruitment health centers were reimbursed by the study and counted as direct costs. Infants with severe ILI warranting hospitalization were admitted to l’Hôpital Gabriel Touré, a public university hospital, and were visited by a study physician daily who accounted for direct and indirect costs. In addition to direct costs to families that were reimbursed through the maternal influenza study, other diagnostic tests  were provided free of charge to some patients through separate ongoing studies when indicated; the costs of these tests were added back into total cost estimates.Pregnant women were recruited during their 3Inpatient episodes included all direct and indirect costs incurred in both the outpatient and inpatient setting for a single ILI episode and were modelled with a log-normal distribution. Outpatient episodes included illnesses with non-zero total costs but with no hospitalization; these were also modelled with a log-normal distribution. All episodes included zero and non-zero costs of outpatient and inpatient episodes and were modelled with an exponential distribution. Statistics were performed using Matlab Version 7.7 .Approval for the research was obtained from the University of Maryland, Baltimore Institutional Review Board; the ethics committee of the Faculté de Médecine, Pharmacie et Odonto-Stomatologie of Mali; and the Ministry of Health of Mali.We performed univariate and multivariate sensitivity analyses to ascertain the major determinants of the cost-effectiveness of maternal influenza immunization. First, each input variable was individually changed by a relative proportion to assess its impact on the CER. Second, we performed a regression tree analysis on 10,000 Monte Carlo simulations that randomly sampled all input variables across their distributions simultaneously. The regression tree algorithm found the threshold value of the single input variable that best divides all simulations into two groups, minimizing the variance in CERs within each group. This process yielded the single parameter and threshold value with the highest predictive power in estimating the CER. The algorithm then performed the same calculation on each respective branch to determine the next most important parameters in predicting the CER and repeated until the number of simulations in each leaf was <10. Finally, the tree was pruned to prevent over-fitting using a 10-fold cross-validation strategy that minimizes generalization error.Under clinical trial conditions, active surveillance likely led to earlier detection of illness, higher healthcare resource utilization, and better health outcomes. For example, low birth weight incidence in the Mali trial was significantly lower than that found in Bamako in Demographic and Health Surveys (DHS) ,16. Thusrd trimester likely maximizes infant protection, but leaves the pregnant woman unimmunized for a larger proportion of the pregnancy. We therefore examined how the interaction of these two attack rates affected the CER.Finally, we performed a two-way sensitivity analysis to look at the impact of changing attack rates on vaccination strategies. Season-to-season and geographical variability in influenza incidence may profoundly affect cost-effectiveness. Additionally, vaccinating earlier in pregnancy might provide greater protection throughout the pregnancy, but may result in less antibody transfer and protection of the infant. Conversely, vaccinating in the 3The model was built and analyzed using the Treeplan, Sensit, and Risksim macros  in MS Excel as well as Matlab Version 7.7 . We calculated 95% confidence intervals (CI) for primary cost data and 95% uncertainty intervals (UI) for Monte Carlo simulations.We captured costs for 188 of 193 laboratory-confirmed influenza (LCI) episodes  and 6,807 of 7,770 influenza-like illness (ILI) episodes . The average total cost per case for 188 episodes of LCI across all populations was $5.84 (CI: $5.08-$6.77), compared to $5.50 (CI: $5.37-$5.63) for 6,807 ILI episodes . There wUnder baseline assumptions, the cost-effectiveness of maternal influenza immunization in Mali was estimated at $857 (UI: $188-$2358) per disability-adjusted life year (DALY) saved . AdjustiGiven the high sensitivity of our model to programmatic costs of vaccinating pregnant women and the reality of changing prices for vaccine itself, supplies, personnel, fuel, and other administrative costs, we calculated estimates of expected cost-effectiveness at different total price points for vaccinating pregnant women  and the We studied the interaction of the attack rates of influenza in pregnant women and infants . PerturbMaternal influenza immunization has the potential to be a high-impact, cost-effective intervention for reducing maternal and neonatal morbidity and mortality in low-income countries if programmatic costs are sufficiently low. Cost-effectiveness estimates were significantly lower than per capita GDP in Mali when total cost per vaccinated pregnant woman was less than $0.50, and mean estimates were lower than this threshold at a cost of $1.00 per vaccinated pregnant woman. Maternal immunization’s impact on infant influenza burden provided the greatest benefits in DALYs saved. Cost-effectiveness was most improved in scenarios with increased infant attack rates, hospitalization rates, CFR, and vaccine efficacy.Policy makers seeking to implement maternal influenza vaccine in low-income settings will confront many challenges including deciding how to focus resources on vaccine programs. Efforts aimed at more remote areas, while more costly to administer, would likely yield higher mortality benefits; in populations with poor access to care, mean CER estimates were lower than per capita GDP in Mali at total cost of $1.67 per pregnant woman vaccinated. Logistical considerations such as combination with maternal tetanus vaccine programs or in campaigns for seasonal illnesses in low-income countries (e.g. meningococcus) may also play an important role in the most appropriate timing and location of maternal influenza vaccine programs.To our knowledge, no previous studies have estimated the costs of influenza illness in sub-Saharan Africa . One othAs in all mathematical models, the conclusions from this study are limited by the quality of data informing the parameters. Many parameters including attack rates of influenza, vaccine efficacy, hospitalization rates in infants, and economic impact of influenza are based on two years of primary data collected during a clinical trial in Mali. However, geographic and seasonal variation in influenza severity, vaccine match, and market forces may change these key parameters substantially . AdditioWhile all of these factors decrease the generalizability of this model to future influenza seasons and different low-income countries, we also did not account for several factors that may increase the benefit of maternal influenza vaccine including decreased influenza severity in vaccinated mothers and infants –23, mateMaternal immunization holds promise as a key strategy in the reduction of neonatal and infant mortality through immunologic mechanisms that protect vulnerable young infants too immature to respond effectively to direct vaccination. A maternal influenza immunization program in Mali would likely be highly cost-effective with cost per DALY saved significantly less than per capita GDP if the total cost of the vaccination program could be kept less than $0.50 per pregnant woman vaccinated. Supplemental immunization activities for maternal tetanus vaccine in Pakistan achieved costs as low as $0.40 per dose , and couS1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.