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Adenine nucleotide translocator (ANT) is a mitochondrial protein involved in the exchange of ADP and ATP across the mitochondrial inner membrane. It plays a crucial role in cellular energy metabolism by facilitating the transport of ATP synthesized within the mitochondria to the cytoplasm. The isoform ANT1 predominately expresses in cardiac and skeletal muscles. Mutations or dysregulation in ANT1 have been implicated in various mitochondrial disorders and neuromuscular diseases. We aimed to examine whether ANT1 deletion may affect mitochondrial redox state in our established ANT1-deficient mice. Hearts and quadriceps resected from age-matched wild type (WT) and ANT1-deficient mice were snap-frozen in liquid nitrogen. The Chance redox scanner was utilized to perform 3D optical redox imaging. Each sample underwent scanning across 3-5 sections. Global averaging analysis showed no significant differences in the redox indices (NADH, flavin adenine dinucleotide containing-flavoproteins Fp, and the redox ratio Fp/(NADH+Fp) between WT and ANT1-deficient groups. However, quadriceps had higher Fp than hearts in both groups (p = 0.0004 and 0.01, respectively). Furthermore, the quadriceps were also more oxidized (a higher redox ratio) than hearts in WT group (p = 0.004). NADH levels were similar in all cases. Our data suggest that under non-stressful physical condition, the ANT1-deficient muscle cells were in the same mitochondrial state as WT ones and that the significant difference in the mitochondrial redox state between quadriceps and hearts found in WT might be diminished in ANT1-deficient ones. Redox imaging of muscles under physical stress can be conducted in future.
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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mitochondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1α), shifting metabolism toward glycolysis to drive viral biogenesis but also causing the release of mitochondrial DNA (mtDNA) and activation of innate immunity. To determine whether mitochondrially targeted antioxidants could mitigate these viral effects, we challenged mice expressing human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2 and intervened using transgenic and pharmacological mitochondrially targeted catalytic antioxidants. Transgenic expression of mitochondrially targeted catalase (mCAT) or systemic treatment with EUK8 decreased weight loss, clinical severity, and circulating levels of mtDNA; as well as reduced lung levels of HIF-1α, viral proteins, and inflammatory cytokines. RNA-sequencing of infected lungs revealed that mCAT and Eukarion 8 (EUK8) up-regulated OXPHOS gene expression and down-regulated HIF-1α and its target genes as well as innate immune gene expression. These data demonstrate that SARS-CoV-2 pathology can be mitigated by catalytically reducing mROS, potentially providing a unique host-directed pharmacological therapy for COVID-19 which is not subject to viral mutational resistance.
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Antioxidantes , COVID-19 , Ratones Transgénicos , Mitocondrias , Fosforilación Oxidativa , SARS-CoV-2 , Animales , Ratones , COVID-19/virología , COVID-19/metabolismo , COVID-19/inmunología , COVID-19/patología , Antioxidantes/metabolismo , Antioxidantes/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Pulmón/virología , Pulmón/patología , Pulmón/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Catalasa/metabolismo , Catalasa/genética , Tratamiento Farmacológico de COVID-19 , Modelos Animales de Enfermedad , Inmunidad InnataRESUMEN
Impairment of the central nervous system (CNS) poses a significant health risk for astronauts during long-duration space missions. In this study, we employed an innovative approach by integrating single-cell multiomics (transcriptomics and chromatin accessibility) with spatial transcriptomics to elucidate the impact of spaceflight on the mouse brain in female mice. Our comparative analysis between ground control and spaceflight-exposed animals revealed significant alterations in essential brain processes including neurogenesis, synaptogenesis and synaptic transmission, particularly affecting the cortex, hippocampus, striatum and neuroendocrine structures. Additionally, we observed astrocyte activation and signs of immune dysfunction. At the pathway level, some spaceflight-induced changes in the brain exhibit similarities with neurodegenerative disorders, marked by oxidative stress and protein misfolding. Our integrated spatial multiomics approach serves as a stepping stone towards understanding spaceflight-induced CNS impairments at the level of individual brain regions and cell types, and provides a basis for comparison in future spaceflight studies. For broader scientific impact, all datasets from this study are available through an interactive data portal, as well as the National Aeronautics and Space Administration (NASA) Open Science Data Repository (OSDR).
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Encéfalo , Neuronas , Vuelo Espacial , Animales , Ratones , Femenino , Encéfalo/metabolismo , Encéfalo/patología , Neuronas/metabolismo , Transcriptoma , Neurogénesis , Análisis de la Célula Individual , Ratones Endogámicos C57BL , Transmisión Sináptica , Ingravidez/efectos adversos , Astrocitos/metabolismo , Estrés Oxidativo , Perfilación de la Expresión Génica , MultiómicaRESUMEN
Mitochondrial function is important for both energetic and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations directly impact these functions, resulting in the detrimental consequences seen in human mitochondrial diseases. The role of pathogenic mtDNA mutations in human cancers is less clear; while pathogenic mtDNA mutations are observed in some cancer types, they are almost absent in others. We report here that the proofreading mutant DNA polymerase gamma ( PolG D256A ) induced a high mtDNA mutation burden in non-small-cell lung cancer (NSCLC), and promoted the accumulation of defective mitochondria, which is responsible for decreased tumor cell proliferation and viability and increased cancer survival. In NSCLC cells, pathogenic mtDNA mutations increased glycolysis and caused dependence on glucose. The glucose dependency sustained mitochondrial energetics but at the cost of a decreased NAD+/NADH ratio that inhibited de novo serine synthesis. Insufficient serine synthesis, in turn, impaired the downstream synthesis of GSH and nucleotides, leading to impaired tumor growth that increased cancer survival. Unlike tumors with intact mitochondrial function, NSCLC with pathogenic mtDNA mutations were sensitive to dietary serine and glycine deprivation. Thus, mitochondrial function in NSCLC is required specifically to sustain sufficient serine synthesis for nucleotide production and redox homeostasis to support tumor growth, explaining why these cancers preserve functional mtDNA. In brief: High mtDNA mutation burden in non-small-cell lung cancer (NSCLC) leads to the accumulation of respiration-defective mitochondria and dependency on glucose and glycolytic metabolism. Defective respiratory metabolism causes a massive accumulation of cytosolic nicotinamide adenine dinucleotide + hydrogen (NADH), which impedes serine synthesis and, thereby, glutathione (GSH) and nucleotide synthesis, leading to impaired tumor growth and increased survival. Highlights: Proofreading mutations in Polymerase gamma led to a high burden of mitochondrial DNA mutations, promoting the accumulation of mitochondria with respiratory defects in NSCLC.Defective respiration led to reduced proliferation and viability of NSCLC cells increasing survival to cancer.Defective respiration caused glucose dependency to fuel elevated glycolysis.Altered glucose metabolism is associated with high NADH that limits serine synthesis, leading to impaired GSH and nucleotide production.Mitochondrial respiration defects sensitize NSCLC to dietary serine/glycine starvation, further increasing survival.
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Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.
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Astronautas , Radiación Cósmica , MicroARNs , Vuelo Espacial , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Radiación Cósmica/efectos adversos , Roturas del ADN de Doble Cadena/efectos de la radiación , Traumatismos por Radiación/genética , Traumatismos por Radiación/prevención & control , Masculino , Mitocondrias/efectos de la radiación , Mitocondrias/metabolismo , Mitocondrias/genética , Femenino , AdultoRESUMEN
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
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Leucemia Mieloide Aguda , Mitocondrias , Mitofagia , Proteínas Quinasas , Factores de Empalme Serina-Arginina , Animales , Humanos , Ratones , Sustitución de Aminoácidos , Línea Celular Tumoral , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Mitofagia/genética , Mutación Missense , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Empalme del ARN , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
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To determine the effects of SARS-CoV-2 infection on cellular metabolism, we conducted an exhaustive survey of the cellular metabolic pathways modulated by SARS-CoV-2 infection and confirmed their importance for SARS-CoV-2 propagation by cataloging the effects of specific pathway inhibitors. This revealed that SARS-CoV-2 strongly inhibits mitochondrial oxidative phosphorylation (OXPHOS) resulting in increased mitochondrial reactive oxygen species (mROS) production. The elevated mROS stabilizes HIF-1α which redirects carbon molecules from mitochondrial oxidation through glycolysis and the pentose phosphate pathway (PPP) to provide substrates for viral biogenesis. mROS also induces the release of mitochondrial DNA (mtDNA) which activates innate immunity. The restructuring of cellular energy metabolism is mediated in part by SARS-CoV-2 Orf8 and Orf10 whose expression restructures nuclear DNA (nDNA) and mtDNA OXPHOS gene expression. These viral proteins likely alter the epigenome, either by directly altering histone modifications or by modulating mitochondrial metabolite substrates of epigenome modification enzymes, potentially silencing OXPHOS gene expression and contributing to long-COVID.
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COVID-19 , Mitocondrias , Fosforilación Oxidativa , SARS-CoV-2 , Humanos , COVID-19/metabolismo , COVID-19/genética , COVID-19/virología , Mitocondrias/metabolismo , Mitocondrias/genética , Especies Reactivas de Oxígeno/metabolismo , Epigénesis Genética , Metabolismo Energético , Epigenómica , AnimalesRESUMEN
Prenatal alcohol exposure can produce disruptions in a wide range of cognitive functions, but it is especially detrimental to spatial navigation. In open environments, rodents organize their spatial behaviors around centralized locations, termed home bases, from which they make circuitous and slow locomotor trips (progressions) into the rest of the environment. Open-field behaviors are organized even under darkened test conditions, suggesting a role for self-motion cues (vestibular, motor, etc.). The impact of moderate prenatal alcohol exposure (mPAE) on the organization of spontaneous open-field behaviors under darkened conditions has not been investigated. Here we tested adult female and male rats with mPAE or saccharin control exposure in a circular open field for 30 min in a testing room that was made completely dark. While general locomotion, as measured by reductions in travel distance and increased stop duration, decreased across the test session, the organization of these behaviors, as measured by stop duration, home base establishment, home base stability, progression accuracy, and scaling of peak speeds with progression length, did not differ between mPAE and saccharin control rats. Together, the findings strongly suggest that spontaneous movement organization in relation to self-motion cues remains intact in adult mPAE rats. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
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NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM's potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism.
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Metabolismo Energético , NAD , Ratones , Animales , NAD/metabolismo , Microscopía Fluorescente , Oxidación-Reducción , EnvejecimientoRESUMEN
Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.
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Linfocitos , NAD , Linfocitos/metabolismo , Metaboloma , Transducción de SeñalRESUMEN
The mitigation of climate change and pollution-related hypoxia and anoxia is a growing challenge for coastal communities. Known ocean conservation measures do not show the desired fast results counteracting deoxygenation. The new infrastructure related to the coastal production of renewable energies linked to the production of green hydrogen can provide new possibilities of artificial ocean reoxygenation to mitigate coastal hypoxia, but has to be treated urgently and seriously from different scientific, engineering and socio-economic angles.
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Cambio Climático , Ecosistema , Humanos , Contaminación Ambiental , HipoxiaRESUMEN
BACKGROUND: Although racial and ethnic differences in CPAP adherence for OSA are widely established, no studies have examined the influence of perceived racial discrimination on CPAP usage, to our knowledge. RESEARCH QUESTION: (1) Do Black adults with OSA report experiencing greater amounts of discrimination than non-Hispanic White adults? (2) Is discrimination associated with poorer CPAP adherence over time, independent of self-identified race? (3) Does discrimination mediate the relationship between self-identified Black race and CPAP usage? STUDY DESIGN AND METHODS: In this prospective study, Black and non-Hispanic White adults with OSA initiating CPAP were enrolled from two sleep centers and completed questionnaires including sociodemographics, perceived discrimination, daytime sleepiness, insomnia symptoms, and depressive symptoms. Perceived discrimination was measured using the Everyday Discrimination Scale (EDS). Black and White group comparisons for baseline sociodemographic variables, sleep symptoms, and perceived discrimination were performed with Student t test or χ2/Fisher exact test, as appropriate. A linear regression model was completed with self-identified Black race and EDS total score as the primary independent variables of interest and mean daily CPAP usage at 30 and 90 days serving as the dependent outcomes. This regression modeling was repeated after adjusting for psychosocial variables known to be associated with CPAP usage. EDS total score was explored as a potential mediator of the association between self-identified Black race and mean daily CPAP adherence at 30 and 90 days. RESULTS: The sample for this analysis consisted of 78 participants (31% female, 38% Black) with a mean age of 57 ± 14 years. Sixty percent of the Black adults reported they experienced racial discrimination at least a few times each year. Relative to White adults, Black adults were also more likely to indicate more than one reason for discrimination (27% vs 4%, P = .003). Adjusting for discrimination, self-identified Black race was associated with 1.4 (95% CI, -2.3 to -0.4 h; P = .006) and 1.6 (95% CI, -2.6 to -0.6 h; P = .003) fewer hours of mean daily CPAP usage at 30 and 90 days, respectively. In the fully adjusted model, a 1-unit change in the total discrimination score (more discrimination) was associated with a 0.08-h (95% CI, 0.01-0.15 h; P = .029) and 0.08-h (95% CI, 0.01-0.16 h; P = .045) change in mean daily CPAP usage at 30 and 90 days, respectively. INTERPRETATION: Adults with OSA who encountered racial discrimination experienced greater decrement in CPAP usage than those who did not experience racial discrimination.
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Negro o Afroamericano , Presión de las Vías Aéreas Positiva Contínua , Cooperación del Paciente , Racismo , Apnea Obstructiva del Sueño , Población Blanca , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Presión de las Vías Aéreas Positiva Contínua/psicología , Cooperación del Paciente/etnología , Cooperación del Paciente/psicología , Estudios Prospectivos , Sueño , Apnea Obstructiva del Sueño/etnología , Apnea Obstructiva del Sueño/terapia , Trastornos del Inicio y del Mantenimiento del Sueño/etnología , Trastornos del Inicio y del Mantenimiento del Sueño/terapia , Población Blanca/psicología , Racismo/etnología , Racismo/psicología , Negro o Afroamericano/psicologíaRESUMEN
BACKGROUND: The relationship between perceived social support and continuous positive airway pressure remains understudied among individuals with obstructive sleep apnea. The aim of this prospective cohort study was to determine if baseline perceived social support and subtypes predict regular continuous positive airway pressure use after 1month of therapy. METHODS: Adults with obstructive sleep apnea initiating continuous positive airway pressure therapy were recruited from sleep clinics in New York City. Demographics, medical history, and comorbidities were obtained from patient interview and review of medical records. Objective continuous positive airway pressure adherence data was collected at the first clinical follow-up. RESULTS: Seventy-five participants (32% female; non-Hispanic Black 41%; mean age of 56 ± 14years) provided data. In adjusted analyses, poorer levels of overall social support, and subtypes including informational/emotional support, and positive social interactions were associated with lower continuous positive airway pressure use at 1month. Relative to patients reporting higher levels of support, participants endorsing lower levels of overall social support, positive social interaction and emotional/informational support had 1.6 hours (95% CI: 0.5,2.7, hours; p = .007), 1.3 hours (95% CI: 0.2,2.4; p = .026), and 1.2 hours (95% CI: 0.05,2.4; p = .041) lower mean daily continuous positive airway pressure use at 1month, respectively. CONCLUSION: Focusing on social support overall and positive social interaction particularly, could be an effective approach to improve continuous positive airway pressure adherence in patients at risk of suboptimal adherence.
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Presión de las Vías Aéreas Positiva Contínua , Apnea Obstructiva del Sueño , Adulto , Humanos , Femenino , Persona de Mediana Edad , Anciano , Masculino , Estudios Prospectivos , Cooperación del Paciente , Apnea Obstructiva del Sueño/complicaciones , Apoyo SocialRESUMEN
We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.
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Mitocondrias , Orgánulos , Mitocondrias/metabolismo , Orgánulos/metabolismo , Microscopía/métodos , Potenciales de la Membrana , Colorantes , Colorantes Fluorescentes/químicaRESUMEN
The orphan gene of SARS-CoV-2, ORF10, is the least studied gene in the virus responsible for the COVID-19 pandemic. Recent experimentation indicated ORF10 expression moderates innate immunity in vitro. However, whether ORF10 affects COVID-19 in humans remained unknown. We determine that the ORF10 sequence is identical to the Wuhan-Hu-1 ancestral haplotype in 95% of genomes across five variants of concern (VOC). Four ORF10 variants are associated with less virulent clinical outcomes in the human host: three of these affect ORF10 protein structure, one affects ORF10 RNA structural dynamics. RNA-Seq data from 2070 samples from diverse human cells and tissues reveals ORF10 accumulation is conditionally discordant from that of other SARS-CoV-2 transcripts. Expression of ORF10 in A549 and HEK293 cells perturbs immune-related gene expression networks, alters expression of the majority of mitochondrially-encoded genes of oxidative respiration, and leads to large shifts in levels of 14 newly-identified transcripts. We conclude ORF10 contributes to more severe COVID-19 clinical outcomes in the human host.
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Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.
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Background: Mild obstructive sleep apnoea (OSA) is a common disorder associated with daytime sleepiness and impaired quality of life. Given that adherence to positive airway pressure (PAP) therapy in OSA is suboptimal, alternative strategies are needed particularly for patients with mild OSA. Daytime neuromuscular electrical stimulation (NMES) of the tongue is a new therapeutic modality for mild OSA. The objective of this study was to determine if patients with mild OSA adhere to daytime NMES. Methods: A randomised, sham-controlled, double-masked controlled trial was conducted in 40 patients with mild OSA who received either high-intensity (active) or low-intensity (sham) NMES for 6â weeks. The primary end-point was adherence to therapy. Exploratory outcomes included the respiratory event index (REI) and the Epworth Sleepiness Scale (ESS) score. Results: More than 90% of participants in each arm were adherent to NMES. Exploratory analyses revealed a 32.7% (95% CI 15.5-49.9%) drop in the REI with active NMES, with no significant change in the REI with sham NMES. Improvements were larger in the supine than non-supine REI. Both the apnoea index and hypopnoea index improved with active NMES. Finally, the ESS score improved with active but not with sham NMES. Conclusions: Daytime NMES was well accepted, with a majority using it for the recommended period. NMES of the tongue use was associated with improvements in OSA severity and daytime sleepiness. Additional research is needed to define its role in the treatment armamentarium across the spectrum of OSA severity and in patients who are intolerant to PAP therapy.
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Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2's ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics.
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Aminoácidos , Mutación Missense , Humanos , Mutación , Aprendizaje Automático , Mitocondrias/genéticaRESUMEN
BACKGROUND: Mitochondrial dysfunction is involved in several diseases ranging from genetic mitochondrial disorders to chronic metabolic diseases. An emerging approach to potentially treat mitochondrial dysfunction is the transplantation of autologous live mitochondria to promote cell regeneration. We tested the differential filtration-based mitochondrial isolation protocol established by the McCully laboratory for use in cellular models but found whole cell contaminants in the mitochondrial isolate. METHODS: Therefore, we explored alternative types of 5-µm filters (filters A and B) for isolation of mitochondria from multiple cell lines including HEK293 cells and induced pluripotent stem cells (iPSCs). MitoTracker™ staining combined with flow cytometry was used to quantify the concentration of viable mitochondria. A proof-of-principle mitochondrial transplant was performed using mitoDsRed2-tagged mitochondria into a H9-derived cerebral organoid. RESULTS: We found that filter B provided the highest quality mitochondria as compared to the 5-µm filter used in the original protocol. Using this method, mitochondria were also successfully isolated from induced pluripotent stem cells. To test for viability, mitoDsRed2-tagged mitochondria were isolated and transplanted into H9-derived cerebral organoids and observed that mitochondria were engulfed as indicated by immunofluorescent co-localization of TOMM20 and MAP2. CONCLUSIONS: Thus, use of filter B in a differential filtration approach is ideal for isolating pure and viable mitochondria from cells, allowing us to begin evaluating long-term integration and safety of mitochondrial transplant using cellular sources.