Beyond their role as a cellular powerhouse, mitochondria are emerging as integral players in molecular signaling and cell fate determination through reactive oxygen species (ROS). While ROS production has historically been portrayed as an unregulated process driving oxidative stress and disease pathology, contemporary studies reveal that ROS also facilitate normal physiology. Mitochondria are especially abundant in cardiac tissue; hence, mitochondrial dysregulation and ROS production are thought to contribute significantly to cardiac pathology. Moreover, there is growing appreciation that medical therapies designed to mediate mitochondrial ROS production can be important strategies to ameliorate cardiac disease. In this review, we highlight evidence from animal models that illustrates the strong connections between mitochondrial ROS and cardiac disease, discuss advancements in the development of mitochondria-targeted antioxidant therapies, and identify challenges faced in bringing such therapies into the clinic. Heart disease progression could be tackled by targeting signaling molecules that cause oxidative stress. Jennifer Kwong at Emory University School of Medicine in Atlanta, USA, and co-workers reviewed research into the role of mitochondria and their associated signaling molecules in the development of heart disease. Mitochondria are a major source of reactive oxygen species (ROS), signaling molecules involved in muscle contraction and calcium transfer in the heart, but they also destroy ROS to maintain a balance. Disruption to this balance can lead to elevated ROS, causing DNA and cellular damage, triggering disease. Animal trials using drugs to target mitochondrial ROS show promise in limiting heart disease progression. Further research is needed to determine whether this approach will work in humans and which specific heart problems might benefit from such therapies. However, pathological conditions of unregulated ROS production leading to elevated ROS levels can result in oxidative stress through oxidative damage to DNA, proteins, and lipids, as well as activation of the mitochondrial-permeability transition pore (MPTP), mitochondrial dysfunction, and cell death2. Indeed, dysregulated ROS production and oxidative stress have been implicated in a host of cardiac diseases, including cardiac hypertrophy, heart failure (HF), cardiac ischemia–reperfusion injury (IRI), and diabetic cardiomyopathy (discussed in greater detail in refs 6).Reactive oxygen species (ROS), including the superoxide anion, the hydroxyl radical, and hydrogen peroxide, are critical signaling molecules with important roles in both cardiac physiology and disease. Both cytosolic sources, including NADPH oxidases (NOX), xanthine oxidase, cyclooxygenases, and cytochrome P450 enzymes, and mitochondrial sources, including the respiratory chain, monoamine oxidases (MAOs), p66shc, and NOX4, contribute to the intracellular ROS pool. Under physiological conditions, cardiac ROS signaling regulates heart development and cardiomyocyte maturation, cardiac calcium handling, excitation contraction coupling, and vascular tone  system, and the peroxiredoxin/thioredoxin (PRX/Trx) system. Similar to conditions of unregulated ROS production, an impairment in cellular antioxidant defenses and ROS scavenging can also lead to cardiac dysfunction12, while specific targeting of the mitochondrial ROS pool may be highly beneficial13. In support of a central role for mitochondria-derived ROS in cardiac disease pathogenesis, there has been strong support from in vivo models that link dysregulation of mitochondrial ROS production and/or impairment in mitochondrial ROS scavenging to cardiac dysfunction.In light of the strong connections between ROS and cardiac disease, there has been intense interest in deciphering the mechanisms regulating cardiac ROS production and detoxification, as well as in developing therapies to limit ROS production and enhance ROS detoxification as a means to ameliorate cardiac disease outcomes. Importantly, there has been increasing evidence that strategies targeting global ROS activity, such as trials with systemic administration of the antioxidants vitamin C and vitamin E, have fallen short in their ability to mitigate cardiac diseaseIn this review, we discuss intracellular sources of ROS, the role of oxidative stress in heart disease, evidence linking mitochondrial ROS to cardiac disease, the efficacy of ROS-based therapeutics in the heart, and the emerging frontier of mitochondria-targeted antioxidant therapies and their application to cardiac disease.c oxidoreductase) are recognized as the major sources of ROS within the respiratory chain. For complex I, superoxide production has been suggested to stem from either reduced flavin mononucleotide14 or the N-1a and N-1b iron–sulfur clusters15. For complex III, superoxide has been suggested to be generated at the ubiquinol oxidation site17.The mitochondrial respiratory chain is central to energy production as it couples electron transfer between respiratory chain complexes to proton transport across the mitochondrial inner membrane to generate the electrochemical gradient required for ATP synthesis. In addition to being essential for energy production, the respiratory chain is also the predominant source of intracellular ROS, which are produced as a byproduct of electron transfer Fig. . Althoug18, p66shc contributes to mitochondrial ROS production by oxidizing cytochrome c and stimulating hydrogen peroxide production19.In addition to the respiratory chain, a number of additional mitochondria-localized proteins have been shown to contribute to the mitochondrial ROS pool. These proteins include p66shc,MAOs, and NOX4 Fig. . P66shc The MAO isoforms A and B (MAO-A and MAO-B) have also been identified as important sources of mitochondrial hydrogen peroxide. Localized to the mitochondrial outer membrane, MAOs utilize the cofactor FAD to catalyze the oxidative degradation of monoamines, such as epinephrine and norepinephrine, into hydrogen peroxide and aldehydes. Importantly, both MAO-A and MAO-B are expressed in the heart and, as described in greater detail below, have been shown to play an important role in HF and cardiac IRI.22. NOX4 has been shown to be a source of mitochondrial superoxide and hydrogen peroxide as NOX4 knockdown reduces the production of both ROS species23. More recently, NOX4 activity has been found to be regulated by ATP, suggesting that NOX4 can couple mitochondrial oxidative stress signaling to a cellular energetic state22.NOXs are a family of proteins involved in intracellular ROS production and catalyze the transfer of NADPH to molecular oxygen to generate superoxide and hydrogen peroxide. Of particular interest is NOX4, which partially localizes to the mitochondrial inner membrane in cardiomyocytes and renal cellsTo regulate oxidative stress created by mitochondrial ROS, mitochondria employ an intricate network of ROS scavenging systems that coordinately work to mitigate this stress Fig. . These s. There are three different SOD isoforms  that display distinct cellular localizations, thereby controlling the compartment-specific ROS pools. SOD1, also known as copper-zinc SOD (Cu/Zn SOD), is mainly cytosolic but has also been found to localize to the mitochondrial intermembrane space. SOD2  localizes to the mitochondrial matrix, while SOD3 is found in the extracellular matrix. The control of the mitochondrial localization and activities of SOD1 and SOD2 are critical to mitochondrial ROS scavenging. For SOD1, it has emerged that intermembrane space targeting and activation is dependent on CCS1 (the copper chaperone of SOD1), which aids in enzyme maturation and activation25. In the case of SOD2, it has recently been shown that activity can be regulated by posttranslational acetylation, where acetylation can decrease activity while Sirt3-mediated deacetylation promotes ROS scavenging function26.The first line of defense against mitochondrial ROS is the SODs, which dismutate superoxide into hydrogen peroxide27, suggesting that it can also play a role in controlling the mitochondrial ROS pool.As mentioned above, catalase, the GSH-PX system and PRX/Trx are required for the breakdown of hydrogen peroxide. Catalase is an important peroxisomal antioxidant enzyme that catalyzes the degradation of hydrogen peroxide into water and oxygen. As peroxisomes are home to a number of ROS generating oxidases, peroxisomal catalase is a key component of the intracellular ROS detoxification system. In addition to the peroxisome, catalase has also been detected in heart mitochondria28). In addition to the enzymatic action of GPX, GSH itself is an important nonenzymatic antioxidant due to its ability to directly neutralize the hydroxyl radical and to regenerate the active forms of the antioxidant vitamins E and C29.GSH-PXs 1 and 4  are also localized to mitochondria and utilize reduced glutathione (GSH) to convert hydrogen peroxide into water, thus oxidizing GSH into glutathione disulfide (GSSG). GSSG is then reduced back into GSH by the activity of glutathione reductase, which requires NADPH to regenerate the mitochondrial pool of GSH . Within this family, PRX3 and PRX5 are found in the mitochondria, with PRX3 exhibiting mitochondrial matrix localization and PRX5 localizing to mitochondria, peroxisomes, and the cytosol. PRXs are oxidized during the detoxification of hydrogen peroxide into water and are converted into reduced PRXs by Trxs. Oxidized Trx is in turn reduced by the enzyme Trx reductase using NADPH as a cofactor. Of note, Trx2 is localized to the mitochondria and has been found to play an important role in limiting mitochondrial ROS production and regulating cardiac function11.PRXs comprise a large and conserved family of peroxidases that have the ability to scavenge both hydrogen peroxide and peroxynitrite  has not been as successful40. Given that direct inhibition of the MPTP may pose challenges, targeting MPTP activators such as ROS may be a therapeutic strategy of interest.Mitochondrial dysfunction caused by IRI contributes significantly to myocardial damage41 as well as increased myocardial stresses associated with pressure overload or hypoxia42. HF produces a complex cardiac phenotype involving a combination of cardiomyocyte hypertrophy, fibrosis, arrhythmia, and contractile dysfunction43. NOXs have been implicated in many of these phenotypes, and NOX expression is upregulated in numerous independent studies of HF45. Within the NOX family, NOX2 and 4 are most abundant within the cardiomyocyte. Angiotensin II has been shown to upregulate ROS production in cardiomyocytes through NOX2, causing cardiomyocyte hypertrophy, and profibrotic and proinflammatory changes that lead to remodeling within the cardiovascular system46. While NOX2 causes cardiac hypertrophy through angiotensin II, NOX4, which partially localizes to the mitochondria, is associated with cardiac hypertrophy due to increased pressure overload from myocardial stresses in the failing human heart. While NOX4 is found to increase myocardial angiogenesis and protect against contractile dysfunction in HF42, recent evidence suggests that NOX4 in failing heart models undergoes alternative splicing, which may explain its detrimental involvement in HF47. Other studies have shown that NOX4 depletion decreased mitochondrial swelling and cytochrome c release and decreased mitochondrial DNA (mtDNA) damage. These contradictory results leave many uncertainties related to the role of NOX4 in HF.While IRI-related ROS injury is acute and occurs in a matter of hours to days, ROS-related effects in HF are more chronic. ROS production in HF has been attributed to chronic neurohormonal activation and consequent upregulation of angiotensin IIThe role of NOXs in HF continues to draw widespread interest due to their potential as mitochondrial-specific therapeutic targets for disease modulation.48, while the remaining energy is generated by carbohydrates. Healthy cardiomyocytes use their energy sources flexibly to match ATP demands and oxygen availability. Fatty acid β oxidation (FAO), which mainly takes place in the mitochondria and peroxisomes, requires high oxygen availability and is inefficient in times of high ATP needs. In diabetic hearts, due to high circulating and stored fatty acids, FAO continues to be a predominant source of ATP, and there is a relative loss in energy contribution from carbohydrates and glucose during periods of high ATP demand48. The loss in flexibility between energy sources causes reduced cardiac efficiency and contractile dysfunction, which is a hallmark of diabetic cardiomyopathy. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that largely modulate glucose and fatty acid metabolism and have been implicated in diabetic cardiomyopathy. Animal studies suggest that elevated levels of PPAR-γ and decreased levels of PPAR-α in diabetic cardiomyopathy alter glucose transportation and increase fatty acid accumulation within the cardiomyocyte, thereby altering FAO kinetics49. Furthermore, PPAR contributes to a decrease in mitochondrial oxidative phosphorylation and causes downstream effects that regulate mitochondrial ultrastructure and number. An increase in FAO causes increases in mitochondrial inner membrane potential and stimulates ROS51, which further causes oxidative damage of mitochondrial proteins and DNA, making them less efficient in energy metabolism. Mitochondria in diabetic cardiomyopathy are further compromised due to chronic upregulation of uncoupling proteins found within the inner mitochondrial membrane that adversely affect mitochondrial energetics and calcium stores52. The cumulative damage to mitochondria caused by metabolic derangements in diabetes further upregulates ROS production that promotes apoptosis, as suggested by animal studies53. Over time, apoptotic cardiomyocytes are replaced by fibroblasts, and this change in tissue composition has been implicated in HF with preserved ejection fraction54.Cardiomyopathy in diabetic patients is associated with metabolic abnormalities related to high levels of circulating fatty acids as well as elevated fatty acid stores within cardiomyocytes. Up to 70% of the ATP generated by the healthy heart is produced through the oxidation of fatty acids56. Of note, mutator animals develop accelerated age-associated cardiomyopathy, characterized by increased hypertrophy, ventricular dilation, fibrosis, and cardiac dysfunction56. While this accelerated aging observed in the mutator animals has been suggested to be due in part to mitochondrial dysfunction induced by the accumulation of mtDNA lesions57, mitochondrial oxidative damage has also been suggested to play a role58. The contribution of mitochondrial ROS to the cardiac aging phenotype is underscored by the observations that the treatment of mutator mice with SkQ1 (10-(6′-plastoquinonyl)decyltri-phenylphosphonium cation, a mitochondrial antioxidant) improves cardiac mitochondrial ultrastructure58 and that enhancing mitochondrial ROS scavenging with a mitochondria-targeted catalase ameliorates the exaggerated aging-associated cardiomyopathy56.The central role of mitochondrial ROS and heart disease is highlighted by a number of genetic models in which the modulation of either mitochondrial ROS production pathways or mitochondrial ROS scavenging systems has a significant impact on cardiac physiology and the development of cardiac disease  model of chronic pressure overload-induced cardiac hypertrophy and HF, inhibition of MAO-A with clorgyline (a MAO-A-specific inhibitor) reduces oxidative stress, hypertrophy, and cardiomyocyte death70. Cardiac-specific NOX4-knockout mouse models have demonstrated that mitochondrial NOX4 is integral to mitochondrial dysfunction, cardiomyocyte apoptosis, and left ventricular dysfunction through increased production of superoxide, leading to oxidative modification of mitochondrial proteins, which contributes to cardiac dysfunction20. While there have been conflicting reports of the impact of NOX4 in mouse models of cardiac pressure overload42, the coincident upregulation of NOX4 with cardiac disease is highly suggestive that NOX4-mediated ROS production contributes to disease pathogenesis.As mentioned above, NOX4 partially localizes to the mitochondria and can be a source of mitochondrial superoxide. NOX4 has come into focus in the setting of cardiac disease, as NOX4 expression has been found to be elevated in HF, atrial fibrillation, and atherosclerosis7. While mice heterozygous for the loss of MnSOD are phenotypically normal at baseline, cardiac mitochondria from these animals are sensitized to MPTP activation and ROS-induced cardiomyocyte death71. In contrast to the deleterious effects of MnSOD depletion, cardiomyocyte overexpression of MnSOD is protective in the OVE26 mouse model of type 1 diabetes and diabetic cardiomyopathy72.The dysregulation of mitochondrial ROS scavenging systems also provides strong support for a key role of mitochondrial ROS in cardiac disease. The pathological consequences of impaired MnSOD-mediated ROS detoxification are clear examples. MnSOD homozygous knockout mice are neonatal lethal, dying by postnatal day 10 with dilated cardiomyopathy characterized by left ventricular dilation, cardiomyocyte hypertrophy, and fibrosis9, suggesting that the loss of Gpx1 potentiates cardiac disease. However, the effects of Gpx1 ablation on IRI may be more complex, as it has been observed that Gpx1 ablation sensitizes male mice to IRI, but female mice are protected through a pathway that may involve enhanced nitrate consumption and nitric oxide production73. Nonetheless, mice overexpressing Gpx1 display enhanced survival following myocardial infarction, and while infarct size was not reduced, Gpx1-overexpressing mice display enhanced ventricular function with less cardiomyocyte death74.Reduction in GSH-PX activity has also been found to impact cardiac disease progression. Mice lacking Gpx1 display increased cardiac hypertrophy in response to prolonged angiotensin II infusion and display increased infarct sizes following IRI10, and mice with cardiomyocyte-specific deletion of Trx2 develop dilated cardiomyopathy with a severe decline in cardiac contractility and early mortality11. Conversely, enhancing this system through the overexpression of Prx3 resulted in mice with reduced hypertrophy, fibrosis, and cardiomyocyte death with increased survival following myocardial infarction75.Similar to the reduction in GSH-PX activity, the disruption of the PRX/Trx-mediated ROS scavenging through the depletion of Trx2 neonatal rat ventricular cardiomyocytes causes cardiomyocyte hypertrophy76. These protective effects of mitochondrial catalase expression extended to the TAC model of cardiac hypertrophy and HF. Importantly, alterations of the mitochondrial proteome due to chronic pressure overload are attenuated with mitochondrial catalase-mediated ROS detoxification77. Together, these studies provide strong support for a central role for mitochondrial ROS in potentiating cardiac disease and for mitochondrial ROS scavenging as an avenue for therapy.Finally, the cardioprotective effects of mitochondria-specific ROS scavenging were directly demonstrated by studies comparing the effects of enforced cytosolic versus mitochondrial expression of catalase. While both cytosolic and mitochondria-targeted catalase effectively reduced oxidative stress, only mice overexpressing mitochondrial catalase  displayed significantly reduced cardiac hypertrophy and fibrosis with improved cardiac function in the angiotensin II model of cardiac hypertrophyN-acetylcysteine (NAC), on the treatment of heart disease. However, studies examining the impact of vitamin C and vitamin E with expanded patient cohorts do not support any beneficial effects of antioxidant vitamin supplementation, and the confirmation of the effects of NAC on larger patient cohorts is still outstanding , treatment with these supplements had no effect on preventing the future occurrence of cardiovascular events82. Moreover, a host of additional studies also conclude that vitamin C and/or vitamin E, given intravenously or orally, has little to no effect on the occurrence of cardiovascular events in healthy women83 and male smokers85. Moreover, instead of being protective, vitamin C treatment may worsen muscle metabolism in chronic HF patients86. Finally, a meta-analysis of 15 independent randomized controlled trials encompassing 188,209 patients assessing the effect of vitamin antioxidant supplementation and the prevention of cardiovascular events revealed that vitamin administration had no effect on the incidence of cardiovascular events12. Together, these results indicate that the global antioxidants vitamin C and E have little to no effect on heart disease.Vitamin E is a lipid-soluble antioxidant that can terminate lipid peroxidation chain reactions by stabilizing lipid peroxyl radicals, while vitamin C is a water-soluble radical-scavenging antioxidant with the ability to regenerate vitamin E in cellular membranes. Clinical trials using these antioxidant vitamins to decrease oxidative stress in cardiovascular disease have yielded inconsistent results. Initial studies, with numbers of participants ranging from 10 to 55, evaluating the effect of vitamin C in chronic HF showed promising results87. In addition to ROS scavenging, NAC has the ability to potentiate the vasodilation, hemodynamic, and anticoagulation effects of nitroglycerin;88 thus, the combinatorial use of NAC together with nitroglycerin has been a strategy of great interest. Preliminary trials assessing the effect of NAC together with nitroglycerin and streptokinase have been promising, with patients given NAC displaying reduced oxidative stress and enhanced ventricular function90. Moreover, the subsequent NACIAM  trial, evaluating the use of high-dose NAC in combination with low-dose nitroglycerin in ST-segment elevation myocardial infarction patients with percutaneous coronary intervention, also showed that combined NAC and nitroglycerin treatment could significantly reduce infarct size and increase myocardial salvage91. While the success of these preliminary clinical trials with limited number of patients is encouraging, larger multicenter clinical trials are needed to determine if the beneficial effects of combined NAC and nitroglycerin treatment can be replicated and broadly applied.NAC is a thiol-containing antioxidant that has the ability to regenerate intracellular antioxidant pools, as NAC deacetylation produces cysteine, which is a precursor for reduced GSH. Initial small trials evaluating the efficacy of intravenous NAC on acute MI and ischemic heart disease have shown promise. In the 30 patient ISLAND (Infarct Size Limitation: Acute NAC Defense) trial to assess the effect of NAC on reperfusion injury, NAC administration in combination with streptokinase-mediated recanalization reduced infarct size and improved left ventricular EFIn light of the limited success of general antioxidants in mitigating heart disease and the strong preclinical animal studies supporting the idea that limiting ROS production or enhancing ROS scavenging in the mitochondrial compartment can be highly beneficial to the heart, in recent years, considerable effort has been directed towards developing mitochondria-targeted antioxidants as pharmacological agents to ameliorate disease. MitoTEMPO, mitoQ, and SS-31/MPT-131 are some of the mitochondria-targeted agents that have garnered the most interest.1-oxy) conjugated to the lipophilic triphenylphosphonium cation (TPP+) that facilitates the membrane potential-dependent accumulation of the compound into the matrix92. MitoTEMPO detoxifies superoxide by cycling between its nitroxide and oxoammonium forms, as well as oxidizing ferrous iron to limit hydroxyl radical formation92. MitoTEMPO has been studied in a wide range of animal models of cardiac disease. In the guinea pig ascending aortic constriction/β-adrenergic stimulation model of HF and sudden cardiac death, mitoTEMPO effectively reduced ROS production in both mitochondrial and cytosolic compartments in failing myocytes, and chronic administration prevented HF93. Importantly, the administration of mitoTEMPO after the onset of cardiac hypertrophy was protective, suggesting that cardiac remodeling can be reversed and that mitochondrial ROS scavenging can be deployed as a therapy following the onset of disease93. The protective effects of mitoTEMPO extend to the murine TAC model of chronic pressure overload as long-term mitoTEMPO administration markedly improves both mitochondrial respiratory chain function and cardiac contractile performance94. Finally, mitoTEMPO has also been shown to be beneficial in the setting of diabetic cardiomyopathy, as it has been demonstrated to reduce hypertrophy and preserve cardiac function in the streptozotocin and db/db mouse models of type 1 and type 2 diabetes95.MitoTEMPO is a mitochondria-targeted SOD mimetic that consists of piperidine nitroxide  is a mitochondrial matrix-targeted ubiquinone appended to the TPP+ cation and mitochondrial membrane potential for mitochondrial targeting, SS-31 (d-Arg-2′6′-dimethylTyr-Lys-Phe-NH2) and its acetate salt MTP-131  are mitochondria-targeted antioxidant peptides that harbor alternating aromatic-cationic motifs that facilitate high solubility and membrane permeability100. SS-31/MPT-131 is unique in its ability to accumulate at the mitochondrial inner membrane in a membrane potential-independent fashion, thus allowing the possibility of this compound to act on both functional and diseased mitochondria. At the inner membrane, SS-31 binds to cardiolipin, which is an inner membrane-specific phospholipid with a central role in the regulation of cristae architecture, mtDNA nucleoid distribution, respiratory chain complex integrity, and supercomplex organization101. SS-31 has been shown to specifically modulate cardiolipin’s interaction with cytochrome c102. While the main function of cytochrome c in the respiratory chain is to shuttle electrons between complex III and complex IV, cytochrome c can also function as a peroxidase and thereby mediate mitochondrial oxidative damage. This balance between the electron carrier and peroxidase activities of cytochrome c is controlled by cardiolipin. Hydrophobic binding between cardiolipin and cytochrome c facilitates a partial unfolding of cytochrome c that promotes peroxidase activity. SS-31 works by binding to cardiolipin and disrupting the cardiolipin–cytochrome c interaction, thereby promoting the function of cytochrome c as a respiratory chain electron carrier103.Unlike mitoTEMPO or mitoQ, which require the TPP104. In vivo, SS-31 has shown incredible promise to limit myocardial damage and promote cardiac function in animal models of ischemic injury and HF. In the context of cardiac IRI, the administration of SS-31 either at the onset of ischemia or even just prior to reperfusion significantly reduces infarct size in mouse, rat, guinea pig, and rabbit models107. Additionally, SS-31 has been demonstrated to reduce the extent of microvascular damage in an ex vivo rabbit model of IR, collectively supporting the concept that mitochondrial ROS plays a significant role in reoxygenation-induced myocardial damage and that SS-31 is cardioprotective. In the setting of HF, SS-31 restricts cardiac hypertrophy, reduces fibrosis, improves cardiac function in mice subjected to TAC108, and, importantly, profoundly reduces the degree of mitochondrial ultrastructural and proteomic changes that occur108. Similar to the cardioprotective effects observed in mice, Elamipretide was highly protective in a canine model of microembolism-induced HF. Dogs subjected to a 3-month chronic daily Elamipretide regimen following HF onset displayed decreased ROS burden, enhanced cardiac EF, a normalization of serum biomarkers of HF, as well as preserved mitochondrial function109. Collectively, these animal studies suggest that mitochondrial oxidative damage is a fundamental pathogenic mechanism in both IRI and HF and that targeting mitochondrial oxidative stress through cardiolipin would be of great therapeutic benefit.SS-31 has been widely studied, and in vitro, it has been found to limit mitochondrial ROS production, reduce oxidative damage, and improve mitochondrial bioenergetics13. In this study, patients were treated with a single intravenous infusion of Elamipretide, and cardiac function was evaluated for 24 h following drug administration13. Importantly, patients treated with high-dose Elamipretide displayed a significant decrease in left ventricular end diastolic and end systolic volumes13, suggesting that Elamipretide can improve cardiac function in the context of HF. However, in the EMBRACE STEMI multicenter phase IIa trial to assess the effect of MPT-131 in patients with ST-elevated myocardial infarction, MPT-131, while safe and well tolerated in patients, had no significant impact on infarct size or ventricular function110. Taken together, the safety profile of SS-31-based compounds in patients, together with the preliminary success with acute treatment in the setting of HF with reduced EF, suggest that antioxidant peptide-mediated mitochondrial ROS scavenging holds promise for use in the clinical setting. However, the EMBRACE STEMI trial highlights the challenges of translating drug efficacy in animal studies to benefits in patients. Elucidating the optimal dosing, timing, and duration of drug administration and understanding whether these parameters are specific for different types of cardiac disease will need to be at the forefront of research efforts as we move to bring mitochondria-targeted antioxidant therapy from the bench to the bedside.In light of the promising results of drug-based mitochondrial ROS scavenging in animal models of heart disease, there has been intense interest in translating these benefits into patients. SS-31 has garnered the most interest due to its efficacy in numerous preclinical animal trials. However, the translation of the benefits of SS-31 into use in human patients has proven challenging. Recently, Elamipretide was tested in a double-blind and placebo-controlled trial in patients presenting with HF with reduced EFIn recent decades, significant progress has been made in developing therapeutics to preserve mitochondrial integrity and attenuate oxidative stress in heart disease. Many of these potential treatments show great promise in vitro, and one of the greatest challenges we now face is translating efficacy in preclinical animal studies into therapies for ROS-mediated cardiomyopathies in human patients. In making the transition from the bench to the bedside, our understanding of some key issues will need to be expanded. Critically, we will need to understand what types of heart disease would most benefit from mitochondrial antioxidant therapy and if there are some disease modalities that would benefit from global versus mitochondria-targeted strategies. In addition, in the design of therapeutic regimens, it will be essential to know at what stages of disease development mitochondrial antioxidant therapies should be optimally deployed. Moreover, as the mitochondrial ROS pool plays a significant role in disease pathogenesis, the development of compounds with improved mitochondrial targeting and enhanced antioxidant activity will be key to oxidative stress-based approaches to treating human cardiovascular diseases. In addition to new and more efficacious mitochondrial ROS scavenging compounds, given that ROS can play a role in both physiological and pathological signaling, understanding the differences between these two modes and developing strategies to mitigate pathological ROS damage while preserving physiological ROS signaling will be an important next challenge.


China) that has experienced dramatic changes in the past five decades. These changes include: (1) a trophic state change from mesotrophy to hypertrophy; (2) dense blooms of cyanobacteria during every summer from the 1970s to 1984; (3) a cessation of blooms starting in 1985, with no recurrence; and (4) an increase, coincident with bloom declines, in the production of silver and bighead carp (filter-feeders) by more than tenfold. There are several possible explanations for the disappearance of blooms, including changes in nutrient concentrations, increased zooplankton grazing, and increased grazing on algae by fish. The long-term data suggest that changes in nutrients or in zooplankton were not important, but that the remarkably increased fish densities might have played the key role. To test this hypothesis, in situ enclosure experiments were conducted in three years. The main conclusions are as follows: (1) an increased stocking of the lake with carp played a decisive role in the elimination of cyanobacteria blooms; (2) both silver and bighead carp can eliminate cyanobacteria blooms directly by grazing; (3) zooplankton cannot suppress the blooms; and (4) the lake still is vulnerable to the outbreak of blooms, should fish grazing decline. The critical biomass of carp is approximately 50 g m 3 . The results suggest the applicability of a new food-web manipulation (increased stocking with filter-feeding fish) for controlling cyanobacteria blooms in hypereutrophic lakes. The approach differs from traditional biomanipulation in Europe and North America, where piscivores are added to control planktivores, and this in turn increases zooplankton and decreases algae. The new biomanipulation method is being used or being tested to counteract cyanobacteria blooms in many Chinese lakes such as Lake Dianchi?Xie and Liu: Biomanipulation to Control Cyanobacteria TheScientificWorld (2001) 1, 337-356 in Yunnan Province, Lake Chaohu in Anhui Province, and Lake Taihu in Jiangsu Province. The method has great potential as an important component of an integrated approach to counteract cyanobacteria blooms, especially in lakes where nutrient inputs cannot be reduced sufficiently, and where zooplankton cannot effectively control phytoplankton production.


Ovarian cancer progression involves multifaceted and variable tumor microenvironments (TMEs), from the in situ carcinoma in the fallopian tube or ovary to dissemination into the peritoneal cavity as single cells or spheroids and attachment to the mesothelial-lined surfaces of the omentum, bowel, and abdominal wall. The TME comprises the tumor vasculature and lymphatics , in addition to mesothelial cells, fibroblasts, immune cells, adipocytes and extracellular matrix (ECM) proteins. When generating 3D models of the ovarian cancer TME, researchers must incorporate the most relevant stromal components depending on the TME in question . Such complexity cannot be captured by monolayer 2D culture systems. Moreover, immortalized stromal cell lines, such as mesothelial or fibroblast cell lines, do not always behave the same as primary cells whose response in functional assays may vary from donor to donor; 3D models with primary stromal cells may have more physiological relevance than those using stromal cell lines. In the current review, we discuss the latest developments in organotypic 3D models of the ovarian cancer early metastatic microenvironment. Organotypic culture models comprise two or more interacting cell types from a particular tissue. We focus on organotypic 3D models that include at least one type of primary stromal cell type in an ECM background, such as collagen or fibronectin, plus ovarian cancer cells. We provide an overview of the two most comprehensive current models—a 3D model of the omental mesothelium and a microfluidic model. We describe the cellular and non-cellular components of the models, the incorporation of mechanical forces, and how the models have been adapted and utilized in functional assays. Finally, we review a number of 3D models that do not incorporate primary stromal cells and summarize how integration of current models may be the next essential step in tackling the complexity of the different ovarian cancer TMEs. From tumor initiation to metastasis, intricate and reciprocal interactions between ovarian cancer cells and the stromal components of their surrounding milieu create complex and fluctuating tumor microenvironments (TMEs) ,2. StromOvarian cancer progression involves detachment of cancer cells from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, dissemination into the peritoneal cavity as single cells or spheroids, and attachment to the mesothelial-lined surfaces of the omentum, bowel, and abdominal wall ,9 Figur. OvarianOrganotypic models refer to 3D models, usually containing ECM, that are comprised of two or more cell types to mimic the complex interactions within a tissue. For this review, we focus on organotypic models of the ovarian cancer TME that comprise a 3D culture containing at least one primary stromal cell type in an ECM background, such as collagen or fibronectin, plus ovarian cancer cells. Ovarian cancer cells have a special predilection for the peritoneum and the omentum as sites of metastasis . The outThe 3D organotypic mesothelium model was created to elucidate the role of specific cellular and non-cellular components, namely fibroblasts, HPMCs, and different ECM proteins, in early ovarian cancer metastasis to the omentum . Two keyThis modular mesothelium model has been used in numerous publications that further illuminate the mechanisms involved in early ovarian cancer metastasis ,22,23,241280) on ovarian cancer adhesion/invasion or proliferation was investigated. These assays were followed by confirmatory, counter, and secondary biological assays utilizing the 3D organotypic model of human mesothelium to identify lead compounds. Ultimately, inhibitory activity of the lead compounds on ovarian cancer metastasis was validated in different in vivo xenograft models. A key takeaway from the HTS assays was that many of the compounds screened were active in cancer cells on plastic (>90%), but only a few compounds were effective in the 3D HTS platform (<1%) which directly translated to in vivo activity in xenograft mouse models. Differences in drug response between 2D cultures and 3D organotypic models have also been demonstrated in studies of skin melanoma. These studies reported that treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with either UVB or cisplatin killed melanoma cells in 2D cultures, but only the TRAIL plus cisplatin combination was effective in their layered 3D organotypic skin melanoma spheroid model [The ability to customize the mesothelium model led to its reshaping and utilization in high throughput screening (HTS) assays. Through optimization of parameters such as incubation time, plating sequence, number of ovarian cancer cells, HPMCs, fibroblasts, and ECM, the model was adapted for use in reproducible 384- and 1536- multi-well HTS assays  Figure B. Fully id model ,28,29. TThis mesothelium model is a first step at recapitulating the metastatic microenvironment of ovarian cancer, but it still lacks other in vivo factors such as vasculature, adipocytes, and host immune cells. However, it represents a significantly more complex experimental system than ovarian cancer cells grown in monolayer to analyze the complex mechanisms of tumorigenesis and to potentially identify new therapeutics. Omental cells from different patients in the 3D organotypic cultures reveal a broader picture of donor-to-donor variability in terms of drug response, cellular function and cell signaling. Peritoneal dissemination of ovarian cancer spheroids and their interactions with omental mesothelial cells are not static processes. Hydrodynamic forces generated by increased production of fluid in the peritoneal cavity must be considered in addition to the 3D culture itself. To recreate this aspect of the ovarian cancer TME, Li et al. developed a 3D microfluidic-based platform in which living cells are infused into micrometer-sized chambers . These pA 2018 publication by Carroll et al. added another layer of complexity by investigating the interactions between alternatively activated macrophages (AAMs), mesothelial cells, and ovarian cancer cells in dynamic flow experiments of ovarian cancer cell adhesion . The autCompared with experiments under static conditions, experiments performed under flow conditions provided valuable insights into features of transcoelomic metastasis that cannot be reproduced in standard static cultures, such as increased adhesion under flow conditions . AlthougIdentification of precursor lesions in the fallopian tube fimbria of ovarian cancer patients and BRCA mutation carriers point towards the fimbria as the likely site of origin of high-grade serous ovarian cancer ,31,32, bFallopian tube organoid in vitro models have also been generated from induced pluripotent stem cells (iPSCs). Yucer et al. guided differentiation of iPSC lines into fallopian tube epithelium precursor cells through exposure to BMP4 and WNT4 followed by follistatin, an activin-binding protein that bio-neutralizes members of the TGF-β superfamily . When spOrganoids do not contain any stromal components, but can be incorporated into organotypic culture systems to study the interactions between the organoid cells and the cells of their microenvironment. Taking this concept a step further, one could envision a model in which transformed fallopian tube epithelial cells  are propWhile not a model in the sense that models are constructed, explants of omentum, ovary or fallopian tube pieces cultured in the presence of ovarian cancer cells represent another form of 3D culture. In particular, mouse omentum, ovarian and fallopian tube organ pieces can be cultured for up to two weeks ,38,39. HFor most researchers studying the ovarian cancer TME, access to patient tissue to obtain primary cells will be the limiting factor. A number of valuable 3D models that do not include primary cells have been published and utilized in functional assays. These non-organotypic endeavors to recapitulate the primary ovarian cancer TME in vitro include ovarian cancer cell spheroid cultures on synthetic matrices , on ECM Developing an ideal model for the ovarian cancer TME is not straightforward. Multiple TMEs with varying components mean that a minimum of four models are likely required: in situ carcinoma in the fallopian tube; dissemination in the peritoneal cavity; early metastatic attachment to the mesothelial-lined surfaces of the omentum, bowel, and abdominal wall; and late chemoresistant metastases. Each complete model will first require the comprehensive characterization  of the associated stromal cells and ECM components, the growth factor and metabolite milieu, and, if applicable, the flow rate. Once the components of each TME have been characterized, the primary cells and ECM will need to be isolated, followed by reconstruction of the tissue of interest, with the aid of a bioprinter or synthetic matrices that can be degraded by cells once they form their own ECM architecture. Functionality of the model will then have to be verified. One option for this may be to confirm that the in vitro secreted proteins are analogous to those of the in vivo secretome, for example in terms of drug response or activation of immune cells. Each of these phases of model development is a significant undertaking, and the current models do not come close to the in vivo scenario in terms of the variety of cell types that are involved in each TME.Multiple potential sites of origin and the continuously changing microenvironments at each stage of the disease demand the development of more diverse  and complex 3D models of the ovarian cancer TME. Each phase of progression has a distinct TME with specific components; for example, models of chemoresistance would include cancer-associated fibroblasts , which aCurrently, the mesothelium model is the only 3D organotypic model of the ovarian cancer TME that is utilized by multiple research groups . The mesThe organotypic models discussed here are restrained from reaching their full potential due to the limitations of working with primary tissue, including access to the tissue and the lifespan of the 3D models, the absence of other essential primary features such as vasculature ,51, and Matrices incorporated into the models presented here are purified from other human, rat, or mouse sources. Scaffold properties , includiUltimately, 3D organotypic models of ovarian cancer aim to recapitulate but systematically simplify the in vivo human microenvironment. Our hope is that by increasing the physiological relevance of 3D organotypic microenvironment models of tumor initiation, primary tumor growth, circulating tumor multi-cellular aggregates, different metastatic sites, and chemoresistant ovarian cancer, the clinical significance of ovarian cancer research will be improved. If we want to offer personalized medicine for ovarian cancer patients, we will also need to successfully establish ovarian cancer organoids for biobanking, as observed with the establishment of organoid cultures in breast, bladder and colorectal cancers ,57,58. B


Kinase signaling pathways orchestrate a majority of cellular structures and functions across species. Liver kinase B1  is a ubiquitously expressed master serine/threonine kinase that plays crucial roles in numerous cellular events, such as polarity control, proliferation, differentiation and energy homeostasis, in many types of cells by activating downstream kinases of the AMP-activated protein kinase (AMPK) subfamily members. In contrast to the accumulating evidence for LKB1 functions in nonneuronal tissues, its functions in the nervous system have been relatively less understood until recently. In the brain, LKB1 initially emerged as a principal regulator of axon/dendrite polarity in forebrain neurons. Thereafter, recent investigations have rapidly uncovered diverse and essential functions of LKB1 in the developing and mature nervous system, such as migration, neurite morphogenesis, myelination and the maintenance of neural integrity, demonstrating that LKB1 is also a multifunctional master kinase in the nervous system. In this review article, we summarize the expanding knowledge about the functional aspects of LKB1 signaling in neural development and homeostasis. Caenorhabditis elegans (C. elegans)) was originally identified by screening for genes that regulate the anterior-posterior axis in the C. elegans zygote  promotes the dendrite outgrowth of DA9 neurons as a downstream mediator of the UNC6 (Netrin)-UNC40 (DCC) pathway (Teichmann and Shen, LKB1 in developing Purkinje cells drastically reduces dendritic Robo2 levels and impairs dendrite self-avoidance, and this effect of LKB1 is mediated by its substrate kinases SIK1 and SIK2 (Kuwako and Okano, In addition to its roles in axonal development, LKB1 is also directly engaged in dendrite development. The first evidence for the involvement of LKB1 in dendrite development comes from DA9 motor neurons in In light of the combined evidence, LKB1 may sequentially function in a variety of steps in axon and dendrite development through adopting different downstream effector kinases, such as SAD-A/B, NUAK1 and SIK1/2, in the regulatory pathway of each event, highlighting the central roles of LKB1 signaling in the developmental program that constructs neurite architecture (Figure LKB1 in granule cell precursors results in the failure of their migration because of the decreased number of granule cells in the internal GL, which possibly leads to cortical expansion and the formation of extra lobes (Men et al., Neuronal migration is an essential process to establish the laminar architecture in the mammalian brain. In the developing cerebral cortex, young projection neurons undergo directional radial migration from the ventricular zone along the radial glial fibers toward the pial surface Hatten, . The celLKB1-deficient Schwann cells are unable to complete this metabolic shift, leading to insufficient synthesis of myelin lipids, thereby impairing the myelination of peripheral axons (Pooya et al., LKB1 in Schwann cells disrupts the asymmetric localization of Par-3 and results in hypomyelination (Shen et al., Recent studies demonstrate that LKB1 regulates the Schwann cell-mediated myelination of peripheral axons through two different mechanisms. During postnatal differentiation, Schwann cells undergo drastic metabolic changes from glycolytic to mitochondrial oxidative metabolism, by which they produce the tricarboxylic acid cycle metabolite citrate, a precursor to cellular lipids. C. elegans one-cell embryo, Drosophila oocyte and human intestinal epithelial cell (Watts et al., LKB1 in the developing hair cells causes the malformation of these hair bundles and the subsequent progressive death of outer hair cells, indicating that LKB1 signaling is essential for the planar polarity formation and survival of hair cells (Men et al., Drosophila LKB1 regulates the asymmetric division of larval neuroblasts by controlling uneven cytokinesis as well as the proper localization of the protein complex Bazooka/atypical PKC/Par-6, that mediates the polarized distribution of the cell fate determinants Numb and Prospero, which are primarily sorted into a ganglion mother cell following cytokinesis, thereby establishing the identity of daughter cells (Bonaccorsi et al., LKB1 and its homologs control cell polarity in various types of cells across species, such as the Regulated organelle distribution is a key mechanism to construct highly polarized neural structures. As we mentioned above, LKB1 signaling-dependent regulation of the organelle distribution, including the Golgi apparatus, centrosome and mitochondria, mediates the various steps in neural development, such as axon specification, dendrite morphogenesis, axon branching and migration (Asada et al., 2+ homeostasis plays a key role in regulating neurotransmitter release (Billups and Forsythe, 2+ (Kwon et al., Recent studies have also uncovered the roles of LKB1 in the mature nervous system. LKB1 signaling in hypothalamic proopiomelanocortin neurons regulates the peripheral glucose metabolism through the secretion of α-melanocyte-stimulating hormone (Claret et al., Several studies also demonstrate the crucial roles of LKB1 in maintaining the integrity of the nervous system. The LKB1-AMPK axis is involved in the synaptic aging of retinal neurons observed in old rodents and humans Figure , 2014. ILKB1 deletion in the spinal cord concomitantly with the mid- and ventral brain causes axon degeneration in the thoracic spinal cord (Sun et al., LKB1 is also involved in neuronal survival and axonal integrity. Mitochondrial dysfunction is a central mechanism implicated in numerous neurodegenerative diseases (Nunnari and Suomalainen, Of note, only AMPK has thus far been identified as a downstream effector of LKB1 in a few contexts in the mature nervous system. Thus, the detailed molecular mechanisms of the LKB-dependent control of neuronal homeostasis have yet to be unraveled.To summarize the recent reports that collectively reveal the versatile neural functions of LKB1, LKB1 is now recognized as an essential kinase that supports many aspects of neural development and homeostasis Figure . NeverthKK and HO contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Newcastle disease virus (NDV) is a devastating worldwide poultry pathogen with major implications for global food security. In this study, two highly inbred and genetically distinct chicken lines, Fayoumis and Leghorns, were exposed to a lentogenic strain of NDV, while under the effects of heat stress, in order to understand the genetic mechanisms of resistance during high ambient temperatures. Fayoumis, which are relatively more resistant to pathogens than Leghorns, had larger numbers of differentially expressed genes (DEGs) during the early stages of infection when compared to Leghorns and subsequently down-regulated their immune response at the latter stages to return to homeostasis. Leghorns had very few DEGs across all observed time points, with the majority of DEGs involved with metabolic and glucose-related functions. Proteomic analysis corroborates findings made within Leghorns, while also identifying interesting candidate genes missed by expression profiling. Poor correlation between changes observed in the proteomic and transcriptomic datasets highlights the potential importance of integrative approaches to understand the mechanisms of disease response. Overall, this study provides novel insights into global protein and expression profiles of these two genetic lines, and provides potential genetic targets involved with NDV resistance during heat stress in poultry. Newcastle disease virus (NDV) has become one of the most important avian diseases impacting poultry production and trade worldwide. NDV is a highly contagious viral disease, capable of spreading rapidly among populations of birds and decimating poultry flocks . The WorChanging climate conditions is an additional factor that may impact the host’s resistance and response to disease. Extreme weather events have been found to create more frequent  and inteSeveral studies have investigated the role that host genetics plays in explaining differences in response to NDV without heat treatment ,12,13 inLarge-scale analysis of the proteome can accurately determine the relative abundance of proteins within tissues. Proteomics offers the opportunity to comprehensively investigate the changes in signaling pathways and biological processes that would otherwise be missed by transcriptome sequencing. In addition, quantitative proteomics and the integrative analysis of the transcriptome can provide corroborative evidence for high priority targets to aid in the discovery of unique mechanisms. The objective of this study was to identify candidate genes and proteins involved with the chicken response to NDV infection during heat stress using an integration of proteomic and transcriptomic approaches. We believe this integrated approach will identify novel biomarkers and provide insight into the differences in response to NDV infection during high ambient temperatures between Fayoumi and Leghorn chicken lines.7 EID50 of the La Sota strain of NDV through both ocular and nasal passages. The control group was mock inoculated with 200 μL of 1X phosphate-buffered saline (PBS). At 2, 6, and 10 days post-infection (dpi), four birds per treatment group per genetic line were randomly selected and euthanized with CO2. Lung tissue was harvested and quickly placed into RNAlater  and kept at −80 °C. Additional lung tissue samples were collected at 2 and 6 dpi and snap frozen in liquid nitrogen and then stored at −80 °C until protein extraction. Protein extraction, protein assay, and the in-solution trypsin digestion were performed as reported by Kultz et. al. [The experimental design of this study has been previously described by Wang et. al. 2018 . BrieflyTotal RNA was extracted from the lung of four individuals per treatment and genetic line for each of the three time points. The lung tissue was homogenized in ice cold TRIzol  and processed using a standard phenol:chloroform method and precipitated in 100% ethanol. The RNA pellet was then dissolved into water and treated with DNase I . Strand-specific RNA library preparation was prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. Library validation and quantification was conducted using the Agilent Bioanalyzer High Sensitivity Kit  and Qubit dsDNA HS Assay kit . The 100 base pair, paired-end sequencing was performed on the Illumina HiSeq2500 system with a minimum sequencing depth of 30 million reads per sample. Sequence data have been submitted through the Sequence Read Archive under accession number: SRP155740.2 change in protein abundance and log2 fold change in gene expression was calculated on 585 protein-transcript pairs. Spearman’s correlation coefficient and significance value was determined using R’s cor.test function [Protein identification and quantification was performed as previously described by Kultz et. al. 2016) . Briefly16 . Briefunction  for all Four major factors were included for analysis: condition , line , sex , and time point . Data at each time point consisted of 16 individuals, four per treatment and genetic line. Raw reads from RNA-seq were trimmed using FastQC  to removDifferential gene expression analysis between treated and non-treated individuals was performed within both Fayoumi and Leghorn chickens at 2, 6, and 10 dpi to explore changes in gene expression induced by NDV infection and heat stress. Comparisons between treated and non-treated Fayoumi chickens at 2 dpi identified 122 differentially expressed genes (DEGs) that were up-regulated and 58 DEGs that were down-regulated . The numIngenuity pathway analysis (IPA) of the DEGs identified in the Fayoumi and Leghorn within line comparisons revealed canonical pathways unique to each genetic line A–F. FayoProteins from lung tissue were extracted and quantified from the same birds used for transcriptome analysis at 2 and 6 dpi to identify significantly over or under abundant proteins expressed during the early immune response. The primary goal for protein quantification was to investigate the early stages of infection, and the early proteomic response observed within these timepoints. The 10 dpi timepoint was not investigated for protein quantification due to sample limitations and central focus of the early immune response. Comparing treated to non-treated Fayoumis, we found six and 13 DAPs at 2 and 6 dpi, respectively . DiffereThe strength of the relationship between the change in protein abundance and gene expression changes due to NDV infection and heat treatment were determined for each line and comparison . A totalIL17REL, which is involved in cytokine-mediated signaling, and NOX4, PRDX1, RAB7B, which are part of the phagosome maturation pathway. Interestingly, very few genes were differentially expressed at 6 dpi, however, results from our group show a similar result in the Harderian gland tissue of the same individuals [PGM3, which was significantly up-regulated at both the 2 and 6 dpi timepoints in Leghorns. PGM3 encodes for a protein that mediates glycogen formation and utilization [PGM3 mutants demonstrate immune deficiency [To our knowledge, this study is the first to simultaneously address the molecular events that occur during NDV infection and heat stress using global proteomic and transcriptomic profiling techniques in chickens. Our study investigated the dynamic protein and gene expression changes induced in the lung by NDV at 2, 6, and 10 dpi to understand the host response during heat stress. Differential gene expression analysis between disease states found interesting differences in response patterns when comparing the relatively resistant Fayoumis to the more disease susceptible Leghorns. In the Fayoumi comparisons, there was a large number of DEGs identified at 2 dpi with the majority of the DEGs 122) significantly up-regulated. Notable immune-related genes include ividuals . The 10 ividuals ,12,14. C2 signifilization . Studiesficiency , decreasficiency , and incficiency . Pathways enriched among the DEGs identified provide insight into the potential mechanisms and gene groups utilized in response to NDV infection during heat stress. At 2 dpi, Fayoumi had significant enrichment in two immune-related pathways, serotonin receptor signaling and phagosome maturation. Serotonin receptor signaling is of particular interest due to its role in hormonal regulation of immune function and high expression amongst immune cells with several immunoregulatory functions such as cytokine secretion and T-cell activation . DiffereWhile Fayoumis appears to be activating pathways involved with the immune and antiviral response at 2 and 6 dpi, Leghorns were primarily enriched for metabolic-related pathways such as glycogen degradation II and III, and glucose and glucose-1-phosphate degradation at these timepoints. Leghorns appear to be responding to the effects of infection-induced hyperglycemia at both the 2 and 6 dpi timepoints. During infection, it is common to observe increased glucose utilization during increased periods of fasting. Failure of peripheral glucose clearance can lead to hyperglycemia and result in the dysregulation of the immune response . The indATOX1, SFTPA1 and LL are of interest due to their prominent roles in the innate immune system and defense against respiratory pathogens. Yeast two-hybrid screens found ATOX1 to interact with partners in the TRIM family of proteins related to the immune response [Despite the similarities in pathways and genes enriched in the proteome and transcriptome data, significant correlation was not observed between the two datasets. Many studies have shown evidence that protein and mRNA levels tend to have poor correlation, especially amongst diverse cell types ,35. Thisresponse  and inhiresponse . LL and response . A studyresponse . Howeverresponse ,12. At tresponse . The infIn summary, comparisons within Fayoumis and Leghorns using protein and gene expression profiling identified unique significant pathways and candidate genes involved in the lung’s response to NDV infection during heat stress. The integration of these two methods allowed for global profiling of the birds’ responses and provided corroborative evidence for observations witnessed in the gene expression and protein studies. Additionally, our study identified potentially important sets of genes and proteins which would have been missed through the independent use of either method. The potential roles and underlying molecular mechanisms involved in NDV response during heat treatment require further investigation into the candidate genes and signaling pathways identified.


NR3C1 and the methylation of FKBP5 and HSD11β2; (iii) chronic inflammation and chronic nitrosative and oxidative stress. Finally, it is shown how severe psychological stress adversely affects mitochondrial structure and functioning and is associated with changes in brain mitochondrial DNA copy number and transcription; mitochondria can act as couriers of childhood stress into adulthood.Severe socioeconomic deprivation (SED) and adverse childhood experiences (ACE) are significantly associated with the development in adulthood of (i) enhanced inflammatory status and/or hypothalamic–pituitary–adrenal (HPA) axis dysfunction and (ii) neurological, neuroprogressive, inflammatory and autoimmune diseases. The mechanisms by which these associations take place are detailed. The two sets of consequences are themselves strongly associated, with the first set likely contributing to the second. Mechanisms enabling bidirectional communication between the immune system and the brain are described, including complex signalling pathways facilitated by factors at the level of immune cells. Also detailed are mechanisms underpinning the association between SED, ACE and the genesis of peripheral inflammation, including epigenetic changes to immune system-related gene expression. The duration and magnitude of inflammatory responses can be influenced by genetic factors, including single nucleotide polymorphisms, and by epigenetic factors, whereby pro-inflammatory cytokines, reactive oxygen species, reactive nitrogen species and nuclear factor-κB affect gene DNA methylation and histone acetylation and also induce several microRNAs including miR-155, miR-181b-1 and miR-146a. Adult HPA axis activity is regulated by (i) genetic factors, such as glucocorticoid receptor polymorphisms; (ii) epigenetic factors affecting glucocorticoid receptor function or expression, including the methylation status of alternative promoter regions of  There is a large body of research demonstrating the existence of a significant association between severe socioeconomic deprivation (SED) and adverse childhood experiences (ACE) and the development of enhanced inflammatory status and/or hypothalamic–pituitary–adrenal (HPA) axis dysfunction in adulthood –3. ThereOne concept invoked to explain these observations at least in part is the phenomenon of psychological resilience. Readers interested in an examination of the evidence for and against this explanation are invited to consult recent papers by , 12. AnoRecent data suggest that the extent of allostatic load appears to be determined by defects in the responses of the autonomic nervous system and the HPA axis as well as increasing levels of lipids and glucose and the extent of inflammation, nitrosative and oxidative stress and mitochondrial dysfunction in any given individual , 15  genes and other variations in the genome and epigenome may influence the magnitude and duration of inflammatory responses to cellular stimuli and the subsequent levels of oxidative stress –20. AbnoThese are important observations, as high levels of inflammation and oxidative stress can induce tissue damage and the formation of damage-associated molecular patterns (DAMPs) which can engage with pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) with the production of pro-inflammatory cytokines (PICs) and reactive oxygen species (ROS), thus maintaining and indeed amplifying a chronic state of peripheral inflammation, nitrosative and oxidative stress and mitochondrial dysfunction , 36. TheWhen considered as a whole, there is considerable evidence in support of the proposition that the allostatic load experienced during childhood can inhibit allostasis leading to the development of allostatic overload in adulthood in the context of genetic and or epigenetic vulnerability. There is also growing evidence that the “unholy trinity” of mitochondrial dysfunction, nitrosative and oxidative stress and inflammation may well be the most significant contributors to allostatic load . A detaiMuch of the early research in this area has been focused on the bidirectional interaction between peripheral inflammation resulting from PIC release by APCs in the periphery as part of an evolutionary response to environmental stressors and the neuroendocrine system. Hence, we will consider such research in the first section before going on to consider more recent findings regarding the importance of oxidative stress in the brain and mitochondrial dysfunction. First, however, it seems appropriate to review the quality and quantity of evidence demonstrating an association between the experience of SED and other ACE in childhood and the development of neuropsychiatric, neurodegenerative and autoimmune illnesses in adulthood.There is a large and accumulating body of evidence demonstrating an association between adverse socioeconomic or caregiving experiences in childhood and increased risk of morbidity and mortality from non-communicable chronic health problems in adulthood –44, inclSeveral, mainly cross-sectional, studies have demonstrated that environmental and socioeconomic adversity in childhood is associated with significantly increased levels of systemic inflammation in both childhood –56 and aSocioeconomic status (SES) is a mediator, marker and moderator of diverse other operative variables . ProspecThere is a considerable body of evidence indicating that children from disadvantaged backgrounds experiencing environmental stressors such as low SES and poor housing conditions are significantly more likely to display evidence of persistent HPA axis dysregulation –81. In aHPA axis hypofunction in particular is found in illnesses such as RA –89, SjögBidirectional communication between the immune system and the CNS involving highly complex signalling pathways plays an indispensable role in restraining immune and inflammatory responses in physiological and pathological conditions . This coThe CNS responses to increased immune activity or the development of inflammation are largely mediated via neuronal and hormonal pathways. The neuronal route is mediated via the autonomic nervous system inflammatory reflex, whereby stimulation of the vagus nerve as an example leads to cholinergic interaction with the α7 subunit of the nicotinic acetylcholine surface receptor (α7 nAChR) of splenic macrophages, in turn leading to inhibition of inflammatory cytokine release by these T cells –109  of the hypothalamus via a number of direct or indirect routes (reviewed in ). The PVThe release of GCs results in termination of HPA axis activation, in the absence of pathology, based on negative feedback achieved by the action of GCs on two main receptors, namely the cytosolic GR and the mineralocorticoid receptor (MR) which translocate to the nucleus following ligation , 121. ThBinding of a GC to a GR leads to the dissociation of molecular chaperones from the GR. The GC-GR complex then translocates to the cell nucleus where gene expression is regulated by interaction with glucocorticoid response elements and via protein–protein engagement with a range of other transcription factors, such as activator protein 1 (AP-1), nuclear factor-κB (NF-κB), nuclear factor of activated T cell (NFAT) and signal transducer and activator of transcription (STAT) –127  in childhood and adulthood , 132. ThSeveral research teams have adduced evidence demonstrating that genetic factors strongly influence the intensity and duration of the immune response and the levels of inflammation produced by the activation of enzyme systems such as mitogen activated protein (MAP) kinases and Janus kinase (JAK)/STAT –147. TheThe significance of these findings extend to the observation that prolonged or excessive activation of immune and inflammatory pathways can induce tissue damage, leading to the formation of immunogenic DAMPs , 36. TheDNA methylation levels of CpG islands (DNA sequences with a high frequency of CpG sites) in promoter regions regulates the transcription of cytokine-encoding genes either directly or via regulating the activity of transcription factors such as STAT-1, NF-κB and NFAT-1 , 161. ThIncreased levels of inflammation, in the guise of elevated TNF-α and ROS, induce widespread hypomethylation and activation of a wide range of inflammatory transcription factors including NF-κB , 171. OxmiRNAs are small non-coding RNA molecules with roles in post-transcriptional regulation of gene expression and RNA silencing. Several miRNAs are induced by PICs, ROS, RNS and NF-κB , 174. FuAnother pro-inflammatory miRNA which is upregulated by PICs and NF-κB is miR-146a , 183. UnIn adults, the inflammatory response to socioeconomic and other stressors is determined by genetic and epigenetic factors, with GR polymorphisms and GR methylation status both being involved . GR and NR3C1 or FKBP5 methylation has been established in several recent studies and systematic reviews [NR3C1, FKBP5 and HSD11β2 has been examined in direct relation to an increased risk for the development of any human illness, with the vast bulk of research being focused on the methylation status of alternative promoter regions of NR3C1 [POMC, ACTH, ACTH-R, AVP, CRH, CRH-R1/2 or CRH-BP genes, which are all involved in the regulation of GC levels and activity by the HPA axis, are currently unknown [NR3C1 gene induced by ACE and concluded that the absolute differences in methylation levels are slight, with the majority of authors reporting group differences of less than 5%, which is currently close to the limit of sensitivity of the methylation detection assays utilised [A growing number of research teams have proposed that methylation of genes involved in the regulation of GR function or expression within the HPA axis might be an important vehicle enabling changes in GC regulation in response to prolonged SED and/or ACE , 202. In reviews , 204. Hoof NR3C1 , 202. Th unknown . There iutilised , 206. Thutilised , 208 andutilised .FKBP5 or NR3C1 and increases in protein levels [FKBP5 and NR3C1 methylation in MDD and PTSD patients with a history of SED and or ACE are only seen in people with acknowledged risk genotypes, which does not seem to be the case with SED- and ACE-induced changes in the methylation status of genes involved in the activation and regulation of peripheral immune genes [In addition, it has been suggested that slight changes in gene methylation levels can be the product of failure to stratify results to take account of confounding variables such as age, sex, current SES and cell tissue type , 211. Thn levels  or changn levels . In addine genes , 214–216ne genes . Howeverne genes . For thene genes , 218 andne genes , 220, whne genes . There iThere is a large and accumulating body of evidence confirming the rapid downregulation of HPA axis activity in an environment of chronic systemic inflammation , 223. ThA range of other enzymes and transcription factors upregulated by PICs such as JAK transcription factors, NFAT, AP-1 and NF-κB have the capacity to inhibit GR function , 232. ThAn environment of chronic nitrosative and oxidative stress in the brain disrupts normal HPA axis function via several mechanisms including increased glutamate toxicity and reduced levels of RNA synthesis, RNA stability, mitochondrial function and activity of redox-sensitive kinases. This is a complex area and readers interested in a detailed treatment of this area are invited to consult the work of Spiers and others . GC receNO regulates a plethora of interactions between neuroimmune and neuroendocrine systems in physiologically normal and pathological conditions and neuronal nitric oxide synthase (nNOS) acts as a major modulator of processes governing the development of learning . NO is iThe downstream effects of NO and PICs are different however, and while CRH release, and consequent stimulation of ACTH secretion from the anterior pituitary, involves the synthesis and release of IL-1, IL-6 and TNF-α, NO released in the PVN modulates signal transduction pathways governing corticosterone release from the suprarenal  gland . NO alsoAuthors investigating the effects of social adversity in rodents have reported neuropathological consequences induced in part by increased levels of ROS and RNS coupled with reduced enzymatic and non-enzymatic anti-oxidants in the periphery and in areas of the brain such as the hippocampus forebrain striatum and cortex. Individual findings include reduced levels of superoxide dismutase (SOD) and catalase and widespread disruption of the glutathione (GSH) system such as a reduced GSH/GSSG ratio and depleted levels of glutathione peroxidase (GPx) –252. TheOnce again, accumulating data indicate that increased brain oxidative stress stemming from war experiences is an important factor . For exaThe association between childhood sexual abuse and the development of persistent oxidative stress and inflammation is difficult to assess despite the existence of a plethora of studies investigating the subject. Some authors have reported an increase in both parameters in the short term but not in the long term while other authors have reported no difference compared with age- and sex-matched children with no history of such experiences , 259  isoforms, most notably NOX-2 , 265. ThThe relationship between activated or ramified microglia and upregulated NOX-induced ROS production is of interest as severe or protracted psychological stress and the development of persistent peripheral inflammation known to activate microglia would go some way to explaining the relationship between severe life experiences and oxidative stress in the brain as discussed above , 270 . For exA recent meta-analysis of 23 studies concluded that protracted and/or severe psychological stress is associated with profound and detrimental changes in mitochondrial performance, structural integrity, morphology and dynamics in the periphery and in the brain . The mosThis is of prime importance as the weight of evidence suggests that variations in mtDNA sequences and/or expression influence the neuroendocrine, metabolic, inflammatory and transcriptional response to stress . MoreoveThe mechanisms whereby mitochondrial dysfunction can have detrimental effects on other harbingers of allostatic load are well documented and straightforward. For example, allostasis is an energy requiring process and environmental stressors provoke a significant increase in energy generation (reviewed in ). SimilaEpigenetic regulation of gene transcription, which plays a vital role in enabling allostasis as discussed above, is dependent on metabolites derived from the tricarboxylic acid cycle or produced elsewhere within mitochondria (reviewed in ). Import2+ homeostasis, decreases mitochondrial respiration, damages enzymes of the ETC, reduces membrane potential and increases mitochondrial ROS production [2+ uptake from the cytoplasm, opening the permeability transition pore resulting in the release of cytochrome c and activation of the PI3K/Akt signalling pathway [Mitochondria are also the site of synthesis for GCs and all other steroid hormones and hence mitochondrial dysfunction could lead to significantly reduced GR production leading to a denuded anti-inflammatory response and impaired HPA axis feedback  .This is of importance as research teams have reported that under conditions of psychological stress damaged or dysfunctional mitochondria can release mtDNA and other mitochondrial DAMPs into the cytoplasm and circulation, leading to the development of systemic inflammation, immune activation and inhibition of the HPA axis , 325. ThWe have shown that there is strong evidence that childhood SED and/or ACE is associated with the development of adult HPA axis dysfunction and neuropsychiatric, neurodegenerative and autoimmune illnesses. Important contributors to the allostatic load experience during childhood include mitochondrial dysfunction, nitrosative and oxidative stress and inflammation, which in turn affect the regulation of HPA axis activity, including via epigenetic factors. We have also seen how PICs, RNS and ROS can have other pathological, including neuropathological, consequences and how mitochondria can act as couriers of childhood stress into adulthood. The latter is of importance as changes in mtDNA sequences and/or expression can influence the endocrine, metabolic, inflammatory and transcriptional response to stress. These findings provide pointers to future research and to potential therapeutic interventions.


Chemotherapeutic targeting of microtubules has been the standard of care in treating a variety of malignancies for decades. During mitosis, increased microtubule dynamics are necessary for mitotic spindle formation and successful chromosomal segregation. Microtubule targeting agents (MTAs) disrupt the dynamics necessary for successful spindle assembly and trigger programmed cell death (apoptosis). As the critical regulators of apoptosis, anti-apoptotic BCL2 family members are often amplified during carcinogenesis that can result in MTA resistance. This review outlines how BCL2 family regulation is positioned within the context of MTA treatment and explores the potential of combination therapy of MTAs with emerging BCL2 family inhibitors. Chemotherapeutics aim to exploit unique metabolic differences between cancer and normal cells in an effort to kill cancer cells more rapidly than the surrounding healthy tissue. For this reason, microtubule targeting agents  have been employed in treating a variety of malignancies, including hematologic and solid tumors for decades . MTAs arMicrotubules are polymeric protein assemblies that are involved in many diverse cellular structures and processes. They have key functions outside the cell cycle, including cytoskeleton formation, cell movement, and intra-cellular trafficking. During cell division, microtubules have increasingly critical functions that ensure proper chromosome segregation beginning in interphase and culminating in mitosis. The various functions of microtubules, including both cellular structure and movement, are possible due to their dynamic, hollow, and cylindrical polymeric structure. As polymers, canonical microtubules are formed from 13 protofilament chains composed of both alpha and beta tubulin dimers. This is an evolutionarily conserved structural feature present in all eukaryotic supergroups with the 13 protofilaments providing a linear lattice that runs parallel to the length of the polymer and may allow for direct transport along the microtubule . The seqThe essential unit of both the protofilament and microtubule is the protein tubulin. There are six different eukaryotic families  that comprise the tubulin superfamily. The alpha and beta tubulin families are the major constituents and serve as the primary components that form the microtubule. The gamma tubulin family forms gamma tubulin ring complexes (gamma-TuRC) that serve as templates for microtubule nucleation . The othAlpha and beta tubulin exist in an equilibrium between a soluble pool of tubulin heterodimers and polymerized tubulin that is assembled into microtubules . This eqMTAs were first identified in the middle of the 20th century through natural products research searching for anti-cancer agents . Since tMTAs are a diverse set of compounds, while the primary function of taxanes and vinca alkaloids as either stabilizing or destabilizing divides them into two categories . InteresVinca rosea (Catharanthus roseus) [Both of the vinca alkaloids that ultimately entered the clinic were discovered in the 1950s through analysis of extracts of the subtropical flower  roseus) . Vincris roseus) . Three o roseus) . Althoug roseus) . Vinca a roseus) . In eachTaxus brevifolia (Pacific Yew) bark during a National Cancer Institute screen for natural products with anti-cancer activity [Taxus baccata (English Yew) that was discovered as a result of the immense demand for paclitaxel [Like vinca alkaloids, paclitaxel was discovered in the early 1960s in extracts of activity ,28. Subsactivity ,30. Paclclitaxel . A naturclitaxel . Conveniclitaxel ,31. Paclclitaxel .Unlike vinca alkaloids, paclitaxel and other taxanes predominantly bind polymerized beta tubulin at a separate site within the lumen of the microtubule nestled between parallel protofilaments . InsteadCancer cells have evolved to evade normal regulation of growth and differentiation. Many chemotherapeutics have been identified due to their ability to exploit such changes in cancer cell growth and metabolism. Yet, the same alterations that enable cancer to emerge leads to the proliferation of chemotherapeutic resistant populations that develops either the ability to evade chemotherapy induced death or the ability to remove the chemotherapeutic agents from the cellular environment. As taxanes and vinca alkaloids closely mimic one another’s cellular function, likewise, many of the same mechanisms are employed to gain resistance to both vinca alkaloids and taxanes. These include the induction of multi-drug resistance efflux transporters, e.g., P-glycoprotein, that pump hydrophobic natural products, including vinca alkaloids and taxanes, out of the cell . InteresTo understand the impact of the BCL2 family on MTA efficacy, it is first necessary to review how the BCL2 family regulates apoptosis. BCL2, the eponym of the BCL2 family, was initially observed in the late 1970s through its upregulation due to chromosome 14 translocations in lymphomas ,50. WithThe focal point of BCL2 family regulation centers on the pro-apoptotic effectors, BAK and BAX, which can be induced through upregulation or activation to form homo-oligomers in the mitochondrial outer membrane. These oligomers form pores in the mitochondrial outer membrane, inducing mitochondrial outer membrane permeabilization (MOMP), a necessary event for cytochrome c release and subsequent apoptosis. In a non-stressed cell with basal expression of BAK and BAX, these proteins reside in the cytosol or mitochondrial outer membrane, respectively, in a monomeric and inactive state. Stress-induced upregulation of either BAK or BAX or one of the BH3-only activators , can induce BAK or BAX oligomerization. In either case, oligomerization of BAK and BAX can be held in check by the presence of anti-apoptotic BCL2 family members  that contain all four BH motifs structurally folded in such a way to form a hydrophobic groove or pocket. This pocket binds to amphipathic alpha helical BH3 motifs that are present in both classes of pro-apoptotic BCL2 family members. The affinity of the BH3 helix and the BH3 binding pocket is the hinge between the two poles, pro-apoptotic and pro-survival, within the BCL2 family and the basis for BCL2 regulation of cell fate. The canonical BH3 helix is a structural motif whose primary sequence is capped in the C-terminal direction with aspartic acid . Moving Association of anti-apoptotic BCL2 family proteins with either the BAK or BAX BH3 helix inhibits their oligomerization. The third BCL2 subfamily, the BH3 only proteins, retain only the BH3 motif. Two prevailing models (direct and indirect) were proposed to describe the impact that BH3-only proteins utilize to regulate the BCL2 family ,63. In tThe BCL2 family serve as the gatekeepers of an irreversible intrinsic apoptotic cascade. As critical regulators of cellular viability, the BCL2 family effectively determines cell fate through the interpretation and status of cellular stress. For this reason, many of the functions and capabilities of the BCL2 family have been elucidated through their effects on chemotherapy . The treAs the namesake of the BCL2 family and the template anti-apoptotic family member, the BCL2 protein plays an unexpected role in MTA sensitivity. As an anti-apoptotic protein, it might be expected that elevated levels of BCL2 protein expression would lead to MTA resistance. Interestingly, the opposite is observed in both lung and breast cancer lines where elevated BCL2 protein expression has been shown to increase paclitaxel sensitivity . This isAlthough it shares many functional similarities with its anti-apoptotic homolog, BCL2, the anti-apoptotic BCL2 family member, BCLxL, does not have as complicated a relationship with MTAs as BCL2. Exogenous expression of BCLxL was shown to protect both leukemia and solid tumor cells from paclitaxel-induced cell death ,90. FurtBoth of the anti-apoptotic BCL2 family members BCLW and BFL1/A1 function as much as BCLxL with regard to their impact on MTA sensitivity. Analysis of siRNA knockdown or CRISPR-Cas9 deletion of BCLW resulted in an increase in the rate of paclitaxel-induced cell death , while oThe final anti-apoptotic BCL2 family member, MCL1, has been closely linked with MTA resistance. This involvement in MTA chemoresistance assisted in identifying MCL1′s key role in acting as a mitotic clock and serving as the key anti-apoptotic BCL2 protein during mitosis . During Initial studies to assess the impact of MCL1 on MTA sensitivity highlighted the impact that MCL1 levels have on both sensitivity and resistance to taxanes and vinca alkaloids . LikewisThe tension between the pro- and anti- apoptotic BCL2 proteins dictated by both stoichiometry and binding affinity results in a homeostatic balance within the BCL2 family . CancersThe identification that anti-apoptotic BCL2 family members can regulate cellular sensitivity to MTAs led to a series of studies evaluating how these various inhibitors combine with both taxanes and vinca alkaloids. Thus far, each of these compounds been shown to increase the efficacy of MTAs . Figure Since their identification, MTAs have become the standard of care in treating many cancer malignancies. However, MTAs are not without their problems, namely toxicity toward healthy tissues, including the brain. The BCL2 family, as the key regulators of programmed cell death and crucial mediators for cellular death during mitosis, play a key role in MTA effectiveness. The recent development of small molecules that can specifically target the BCL2 family opened a new avenue for improving cancer therapy and, specifically, MTA effectiveness. The clinical potential of combining MTAs with BCL2 inhibition is only beginning to be explored, yet this limited work already hints at promising new combinations that exploit the ability of BCL2-inhibitors and MTAs to work in synergy to improve targeted cancer cell death, allow for reduced MTA dosage, and result in decreased toxicity. However, knowing when and where to target the BCL2 family individually or as a whole will lead to better combinatorial therapies with existing MTA therapies.