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Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.
Assuntos
Apolipoproteína L1/genética , Variação Genética , Glomerulosclerose Segmentar e Focal/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Podócitos/metabolismo , Negro ou Afro-Americano/genética , Animais , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/fisiopatologia , Homeostase , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/fisiologia , Podócitos/fisiologia , Proteinúria , Triglicerídeos/metabolismoRESUMO
BACKGROUND The phytochemical ingredients of berries have been used in the treatment of various bodily ailments; while their roles in preventing the severity of glaucoma are poorly understood. Hence, the present study was framed to investigate whether ethanolic extracts of Lycium barbarum exerts protection against the onset of glaucoma using cultured PC12 neuronal cells by modulating the expression of extracellular matrix proteins. MATERIAL AND METHODS In order to develop glaucoma like condition in cells, cultured PC12 cells were subjected to 50 and 100 mmHg hydrostatic pressure for 24 hours. The pressure exposed cells were analyzed for the expression of glaucoma markers such as ANGPTL7 and the expressions of extracellular matrix proteins in the presence and absence of L. barbarum, matrix metalloproteinase (MMP)-9 inhibitor, and latanoprost, a current drug for the treatment of glaucoma. RESULTS PC12 cells exposed to hydrostatic pressures (50 and 100 mmHg) increased the expression of glaucoma marker, ANGPTL7. Moreover, results have demonstrated the significant changes in the expression of MMP-2, MMP-9, collagen I, and TGF-ß at the gene level. In contrast, cells pretreated with L. barbarum extracts showed reduced expression of ANGPTL7 and extracellular matrix proteins compared to control. Furthermore, to elucidate the role of MMP-9 in the onset of glaucoma, cells were silenced using MMP-9 inhibitor along with L. barbarum demonstrated a significant reduction in the glaucoma marker ANGPTL7 while improving the expression of caveolin-1 expression in cells subjected to pressure. CONCLUSIONS The extract of L. barbarum protects the cells from intraocular pressure by activating caveolin-1 dependent pathway via inhibition of MMP-9 expression.
Assuntos
Glaucoma/tratamento farmacológico , Lycium/metabolismo , Extratos Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/fisiologia , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
It has been proposed that a loss of bioenergetic capacity of cells contributes to the progressive loss of biological function with age. Aging is associated with loss of mitochondrial cristae membranes and inhibition of ATP production. Despite the many approaches being pursued for improving mitochondrial function, none of them directly targets the electron transport chain to improve ATP production. Recent studies have brought attention to cardiolipin as a unique target for promoting mitochondrial efficiency. Cardiolipin is important for cristae curvatures and is necessary for optimal activity of the respiratory complexes and the assembly of supercomplexes. Here we describe the discovery of a class of cell-penetrating aromatic-cationic tetrapeptides that selectively target cardiolipin and increase coupling efficiency while reducing reactive oxygen species production. These compounds can rejuvenate mitochondrial bioenergetics, remodel mitochondrial cristae structure, repair cellular structure, and restore organ function during aging.
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Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Cardiolipinas/metabolismo , Peptídeos Penetradores de Células/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Regeneração/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , HumanosRESUMO
The innate immune system has been implicated in both AKI and CKD. Damaged mitochondria release danger molecules, such as reactive oxygen species, DNA, and cardiolipin, which can cause NLRP3 inflammasome activation and upregulation of IL-18 and IL-1ß It is not known if mitochondrial damage persists long after ischemia to sustain chronic inflammasome activation. We conducted a 9-month study in Sprague-Dawley rats after 45 minutes of bilateral renal ischemia. We detected glomerular and peritubular capillary rarefaction, macrophage infiltration, and fibrosis at 1 month. Transmission electron microscopy revealed mitochondrial degeneration, mitophagy, and deformed foot processes in podocytes. These changes progressed over the study period, with a persistent increase in renal cortical expression of IL-18, IL-1ß, and TGF-ß, despite a gradual decline in TNF-α expression and macrophage infiltration. Treatment with a mitoprotective agent (SS-31; elamipretide) for 6 weeks, starting 1 month after ischemia, preserved mitochondrial integrity, ameliorated expression levels of all inflammatory markers, restored glomerular capillaries and podocyte structure, and arrested glomerulosclerosis and interstitial fibrosis. Further, helium ion microscopy vividly demonstrated the restoration of podocyte structure by SS-31. The protection by SS-31 was sustained for ≥6 months after treatment ended, with normalization of IL-18 and IL-1ß expression. These results support a role for mitochondrial damage in inflammasome activation and CKD and suggest mitochondrial protection as a novel therapeutic approach that can arrest the progression of CKD. Notably, SS-31 is effective when given long after AKI and provides persistent protection after termination of drug treatment.
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Interleucina-18/fisiologia , Interleucina-1beta/fisiologia , Isquemia/complicações , Rim/irrigação sanguínea , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/prevenção & controle , Regulação para Cima/efeitos dos fármacos , Doença Aguda , Animais , Masculino , Podócitos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity. METHODS: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. RESULTS: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. CONCLUSION: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. GENERAL SIGNIFICANCE: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.
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Obesity is a major risk factor for the development of chronic kidney disease, even independent of its association with hypertension, diabetes, and dyslipidemia. The primary pathologic finding of obesity-related kidney disease is glomerulopathy, with glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis. Proposed mechanisms leading to renal pathology include abnormal lipid metabolism, lipotoxicity, inhibition of AMP kinase, and endoplasmic reticulum stress. Here we report dramatic changes in mitochondrial structure in glomerular endothelial cells, podocytes, and proximal tubular epithelial cells after 28 weeks of a high-fat diet in C57BL/6 mice. Treatment with SS-31, a tetrapeptide that targets cardiolipin and protects mitochondrial cristae structure, during high-fat diet preserved normal mitochondrial structure in all kidney cells, restored renal AMP kinase activity, and prevented intracellular lipid accumulation, endoplasmic reticulum stress, and apoptosis. SS-31 had no effect on weight gain, insulin resistance or hyperglycemia. However, SS-31 prevented loss of glomerular endothelial cells and podocytes, mesangial expansion, glomerulosclerosis, macrophage infiltration, and upregulation of proinflammatory (TNF-α, MCP-1, NF-κB) and profibrotic (TGF-ß) cytokines. Thus, mitochondria protection can overcome lipotoxicity in the kidney and represent a novel upstream target for therapeutic development.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Glomerulonefrite/prevenção & controle , Túbulos Renais Proximais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Capilares/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Glomerulonefrite/etiologia , Túbulos Renais Proximais/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Obesidade/complicações , Oligopeptídeos/farmacologia , Podócitos/efeitos dos fármacosRESUMO
Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.
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Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Isquemia Quente , Trifosfato de Adenosina/biossíntese , Animais , Avaliação Pré-Clínica de Medicamentos , Rim/irrigação sanguínea , Rim/metabolismo , Masculino , Mitocôndrias/metabolismo , Oligopeptídeos/uso terapêutico , Distribuição Aleatória , Ratos Sprague-DawleyRESUMO
Microvascular rarefaction, or loss of microvascular density, is increasingly implicated in the progression from acute ischemic kidney injury to chronic kidney disease. Microvascular dropout results in chronic tissue hypoxia, interstitial inflammation, and fibrosis. There is currently no therapeutic intervention for microvascular rarefaction. We hypothesize that capillary dropout begins with ischemic damage to endothelial mitochondria due to cardiolipin peroxidation, resulting in loss of cristae and the failure to regenerate ATP upon reperfusion. SS-31 is a cell-permeable peptide that targets the inner mitochondrial membrane and binds selectively to cardiolipin. It was recently shown to inhibit cardiolipin peroxidation by cytochrome c peroxidase activity, and it has been shown to protect mitochondrial cristae in proximal tubular cells during ischemia, and accelerated ATP recovery upon reperfusion. We found mitochondrial swelling and loss of cristae membranes in endothelial and medullary tubular epithelial cells after 45-min ischemia in the rat. The loss of cristae membranes limited the ability of these cells to regenerate ATP upon reperfusion and led to loss of vascular integrity and to tubular cell swelling. SS-31 prevented mitochondria swelling and protected cristae membranes in both endothelial and epithelial cells. By minimizing endothelial and epithelial cell injury, SS-31 prevented "no-reflow" after ischemia and significantly reduced the loss of peritubular capillaries and cortical arterioles, interstitial inflammation, and fibrosis at 4 wk after ischemia. These results suggest that mitochondria protection represents an upstream target for pharmacological intervention in microvascular rarefaction and fibrosis.
Assuntos
Injúria Renal Aguda/prevenção & controle , Cardiolipinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nefrite/prevenção & controle , Oligopeptídeos/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Trifosfato de Adenosina/metabolismo , Animais , Citoproteção , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Fibrose , Rim/metabolismo , Rim/fisiopatologia , Rim/ultraestrutura , Masculino , Microvasos/metabolismo , Microvasos/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Dilatação Mitocondrial/efeitos dos fármacos , Nefrite/metabolismo , Nefrite/patologia , Nefrite/fisiopatologia , Oligopeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de TempoRESUMO
Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.
Assuntos
Cardiolipinas/metabolismo , Citocromo-c Peroxidase/metabolismo , Isquemia/metabolismo , Mitocôndrias/efeitos dos fármacos , Oligopeptídeos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Oligopeptídeos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
Although the density peak clustering (DPC) algorithm can effectively distribute samples and quickly identify noise points, it lacks adaptability and cannot consider the local data structure. In addition, clustering algorithms generally suffer from high time complexity. Prior research suggests that clustering algorithms grounded in P systems can mitigate time complexity concerns. Within the realm of membrane systems (P systems), spiking neural P systems (SN P systems), inspired by biological nervous systems, are third-generation neural networks that possess intricate structures and offer substantial parallelism advantages. Thus, this study first improved the DPC by introducing the maximum nearest neighbor distance and K-nearest neighbors (KNN). Moreover, a method based on delayed spiking neural P systems (DSN P systems) was proposed to improve the performance of the algorithm. Subsequently, the DSNP-ANDPC algorithm was proposed. The effectiveness of DSNP-ANDPC was evaluated through comprehensive evaluations across four synthetic datasets and 10 real-world datasets. The proposed method outperformed the other comparison methods in most cases.
Assuntos
Potenciais de Ação , Algoritmos , Redes Neurais de Computação , Análise por Conglomerados , Potenciais de Ação/fisiologia , Neurônios/fisiologia , Humanos , Modelos NeurológicosRESUMO
The burst of reactive oxygen species (ROS) during reperfusion of ischemic tissues can trigger the opening of the mitochondrial permeability transition (MPT) pore, resulting in mitochondrial depolarization, decreased ATP synthesis, and increased ROS production. Rapid recovery of ATP upon reperfusion is essential for survival of tubular cells, and inhibition of oxidative damage can limit inflammation. SS-31 is a mitochondria-targeted tetrapeptide that can scavenge mitochondrial ROS and inhibit MPT, suggesting that it may protect against ischemic renal injury. Here, in a rat model of ischemia-reperfusion (IR) injury, treatment with SS-31 protected mitochondrial structure and respiration during early reperfusion, accelerated recovery of ATP, reduced apoptosis and necrosis of tubular cells, and abrogated tubular dysfunction. In addition, SS-31 reduced medullary vascular congestion, decreased IR-mediated oxidative stress and the inflammatory response, and accelerated the proliferation of surviving tubular cells as early as 1 day after reperfusion. In summary, these results support MPT as an upstream target for pharmacologic intervention in IR injury and support early protection of mitochondrial function as a therapeutic maneuver to prevent tubular apoptosis and necrosis, reduce oxidative stress, and reduce inflammation. SS-31 holds promise for the prevention and treatment of acute kidney injury.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Trifosfato de Adenosina/metabolismo , Antioxidantes/uso terapêutico , Oligopeptídeos/uso terapêutico , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/fisiopatologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/fisiologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Modelos Animais , Oligopeptídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologiaRESUMO
With the development of miniaturization and integration of electronic devices, the conventional manifold microchannels (MMCs) structure has been unable to meet the heat dissipation requirements caused by the rapid growth of internal heat flux. There is an urgent need to design a new heat dissipation structure with higher heat dissipation capacity to ensure the working stability and life of electronic devices. In this paper, we designed a novel manifold dual-microchannel (MDMC) cooling system that embedded the microchannel structure into the manifold microchannel structure. The MDMC not only has good heat dissipation performance that can meet the development needs of electronic equipment to miniaturization and integration, but also has a compact structure that does not increase the overall thickness and volume compared with MMC. The high temperature uniformity and heat transfer performance of MDMC are significantly improved compared to MMC. The Tmax is reduced by 13.6% and 17.5% at the heat flux density of 300 W/cm2 and 700 W/cm2, respectively. In addition, the influence of the inlet-2 velocity and the total microchannels number on the heat transfer performance of the MDMC structure are numerically investigated. The results show that the decrease rate of Tmax and ΔT is about 6.69% and 16% with the increase of inlet-2 velocity from 1.2 m/s to 2.4 m/s and microchannels number from 10 to 48, respectively. At the same time, the best temperature uniformity is obtained when the number of microchannels is 16.
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Surface mount technology (SMT) plays an important role in integrated circuits, but due to thermal stress alternation caused by temperature cycling, it tends to have thermo-mechanical reliability problems. At the same time, considering the environmental and health problems of lead (Pb)-based solders, the electronics industry has turned to lead-free solders, such as ternary alloy Sn-3Ag-0.5Cu (SAC305). As lead-free solders exhibit visco-plastic mechanical properties significantly affected by temperature, their thermo-mechanical reliability has received considerable attention. In this study, the interface delamination of an SMT solder joint using a SAC305 alloy under temperature cycling has been analyzed by the nonlinear finite element method. The results indicate that the highest contact pressure at the four corners of the termination/solder horizontal interface means that delamination is most likely to occur, followed by the y-direction side region of the solder/land interface and the top arc region of the termination/solder vertical interface. It should be noted that in order to keep the shape of the solder joint in the finite element model consistent with the actual situation after the reflow process, a minimum energy-based morphology evolution method has been incorporated into the established finite element model. Eventually, an Improved Efficient Global Optimization (IEGO) method was used to optimize the geometry of the SMT solder joint in order to reduce the contact pressure at critical points and critical regions. The optimization result shows that the contact pressure at the critical points and at the critical regions decreases significantly, which also means that the probability of thermal-induced delamination decreases.
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Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.
Assuntos
Mitocôndrias , Doenças Mitocondriais , Animais , Cardiolipinas/metabolismo , Humanos , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Peptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
AIM: To explore the regulatory mechanism of nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of posterior capsule opacification (PCO). METHODS: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was executed to analyze NEAT1 and microRNA (miR)-26a-5p expression in transforming growth factor-beta 2 (TGF-ß2)-disposed lens epithelial cells (LECs). The proliferation, cell cycle progression, apoptosis, and migration of TGF-ß2-disposed LECs were evaluated. The relationship between NEAT1 or fanconi anemia (FA) complementation group E (FANCE) and miR-26a-5p was verified by dual-luciferase reporter assay. RESULTS: TGF-ß2 induced NEAT1 expression in LECs. NEAT1 inhibition accelerated apoptosis, cell cycle arrest, decreased proliferation, epithelial-mesenchymal transition (EMT), and migration of TGF-ß2-disposed LECs. NEAT1 sponged miR-26a-5p to further regulate FANCE expression. Rescue experiments presented that miR-26a-5p downregulation overturned NEAT1 silencing-mediated impacts on TGF-ß2-disposed LEC biological behaviors. Additionally, FANCE overexpression reversed miR-26a-5p mimic-mediated impacts on TGF-ß2-disposed LEC biological behaviors. CONCLUSION: TGF-ß2-induced NEAT1 facilitates LEC proliferation, migration, and EMT by upregulating FANCE via sequestering miR-26a-5p.
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Songbirds are useful vertebrate study models for vocal learning and memory. The robust nucleus of the arcopallium (RA) receives synaptic inputs from both the posterior and anterior pathways of the song control system in songbirds. Hence, RA plays an important role in the control of singing. RA receives dopaminergic (DArgic) inputs that increase the excitability of RA projection neurons (PNs). However, the effects of DA on excitatory synaptic transmission are yet to be deciphered. In this study, the effects of DA on the excitatory synaptic transmission of the PNs in the RA of adult male zebra finches were investigated using a whole-cell patch-clamp recording. We observed that DA decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The effects of DA were mimicked by the D1-like DA receptor (D1R) agonist, SKF-38393, but not the D2-like DA receptor (D2R) agonist, Quinpirole. Also, the effects of DA were blocked by D1R antagonist, SCH-23390, but not the D2R antagonist, Sulpiride. These results demonstrate that DA modulates excitatory synaptic transmission by acting on D1R in the RA of adult male zebra finches.
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Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Cardiolipinas/metabolismo , Nefropatias Diabéticas/metabolismo , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Cardiolipinas/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Podócitos , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismoRESUMO
OBJECTIVE: Previously, we demonstrated that a novel opiate peptide, 2',6'-dimethyl-tyrosine-D-Arg-Phe-Lys-NH2, provided cardioprotection against myocardial stunning in vivo. We subsequently showed that this peptide targeted mitochondria and can scavenge reactive oxygen species. The objective of this study was to determine the role of opioid versus antioxidant activity in cardioprotection. METHODS: We compared two mitochondria-targeted peptide analogs that lacked opioid activity: SS-31 (D-Arg-2',6'-dimethyl-tyrosine-Lys-Phe-NH2) and SS-20 (Phe-D-Arg-Phe-Lys-NH2). They differ in that only SS-31 has scavenging ability. Rats (n=8/group) were randomized to SS-31, SS-20 or placebo. The drugs (3 mg/kg) or saline was administered intraperitoneally 30 min before ligation of the left anterior descending artery for 60 min, and another dose given intraperitoneally 5 min before reperfusion for 60 min. Study endpoints included myocardial infarct size, cardiac arrhythmia and myocardial lipid peroxidation. RESULTS: The area at risk was similar among the groups. The infarct area/area at risk, however, was significantly smaller in the treatment groups (53.9+/-1.1% in SS-31 group, 47.1+/-1.4% in SS-20 group, versus 59.9+/-1% in the controls, P<0.01). Lipid peroxidation was significantly reduced by both SS-31 and SS-20 treatment. Arrhythmia occurred only during the early period of coronary occlusion and was less frequent and less severe in the peptide treatment groups than in the controls (Lambeth score 5 points, 3 points, versus 13 points in the controls, P<0.05). CONCLUSIONS: This study shows that pretreatment with both SS-31 and SS-20 significantly reduced myocardial lipid peroxidation and infarct size in ischemia-reperfusion injury, and suggests that the cardioprotective properties of 2',6'-dimethyl-tyrosine-D-Arg-Phe-Lys-NH2 was primarily mediated by its antioxidant properties. As SS-20 does not scavenge reactive oxygen species, it most likely reduces reactive oxygen species production during ischemia-reperfusion.
Assuntos
Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Oligopeptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Reperfusão Miocárdica , Ratos , Ratos Sprague-DawleyRESUMO
We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.
Assuntos
Carboidratos/química , Animais , Biotecnologia/métodos , Colódio/química , Dextranos/química , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Inulina/química , Microscopia de Fluorescência , Polissacarídeos/química , TemperaturaRESUMO
Previous studies suggested that neurotrophins play a role in the diabetic retinopathy (DR). We therefore evaluated the role of plasma brain derived neurotrophic factor (BDNF) levels in Chinese type 2 diabetic patients with and without diabetic retinopathy (DR). Plasma levels of BDNF were determined in type 2 diabetic patients (N=344). At baseline, the demographical and clinical data were taken. Multivariate analyses were performed using logistic regression models. Receiver operating characteristic curves (ROC) was used to test the overall predict accuracy of BDNF and other markers. Diabetic patients with DR and vision-threatening diabetic retinopathy (VTDR) had significantly lower BDNF levels on admission (P<0.0001 both). BDNF improved the area under the receiver operating characteristic curve of the diabetes duration for DR from 0.76 (95% confidence interval [CI], 0.71-0.82) to 0.89 (95% CI, 0.82-0.95; P<0.01) and for VDTR from 0.84 (95% CI, 0.78-0.92) to 0.95 (95% CI, 0.90-0.98; P<0.01). Multivariate logistic regression analysis adjusted for common risk factors showed that plasma BDNF levels≤12.4 ng/mL(1(rd) quartiles) was an independent marker of DR (OR=3.92; 95%CI: 2.31-6.56) and VTDR (OR=4.88; 95%CI: 2.21-9.30). The present study demonstrated that decreased plasma levels of BDNF were independent markers for DR and VDTR in Chinese type 2 diabetic patients, suggesting a possible role of BDNF in the pathogenesis of DR complications.