bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒06‒27
forty-eight papers selected by
Catalina Vasilescu
University of Helsinki
  1. Mitochondrial function in development and disease.
  2. Myopathic mitochondrial DNA depletion syndrome associated with biallelic variants in LIG3.
  3. Exploiting hiPSCs in Leber's Hereditary Optic Neuropathy (LHON): Present Achievements and Future Perspectives.
  4. Mitochondrial metabolism regulates macrophage biology.
  5. Parp mutations protect from mitochondrial toxicity in Alzheimer's disease.
  6. Light-inducible deformation of mitochondria in live cells.
  7. Mitochondrial DNA disorders: From pathogenic variants to preventing transmission.
  8. A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients.
  9. Mitochondrial myopathy with a unique presentation of facial and bulbar muscle weakness caused by single large scale mitochondrial DNA deletion.
  10. Mitochondrial disease in adults: recent advances and future promise.
  11. Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.
  12. Association of mitochondrial variants and haplogroups identified by whole exome sequencing with Alzheimer's disease.
  13. The mitochondrial permeability transition pore: an evolving concept critical for cell life and death.
  14. Mitochondrial Disorders.
  15. Leber's Hereditary Optic Neuropathy Arising From the Synergy Between ND1 3635G>A Mutation and Mitochondrial YARS2 Mutations.
  16. Mitochondrial Ca2+ oscillation induces mitophagy initiation through the PINK1-Parkin pathway.
  17. General anesthesia with remimazolam in a patient with mitochondrial encephalomyopathy: a case report.
  18. Occult primary white matter impairment in Leber's hereditary optic neuropathy.
  19. Mechanistic perspectives on differential mitochondrial-based neuroprotective effects of several carnitine forms in Alzheimer's disease in vitro model.
  20. Advances in the mechanism and treatment of mitochondrial quality control involved in myocardial infarction.
  21. Blood Mitochondrial DNA Copy Number: What Are We Counting?
  22. Mitochondrial NAD(P)+ transhydrogenase: from molecular features to physiology and disease.
  23. Loss-of-function variants in DNM1 cause a specific form of developmental and epileptic encephalopathy only in biallelic state.
  24. Coordinated pyruvate kinase activity is crucial for metabolic adaptation and cell survival during mitochondrial dysfunction.
  25. Single-Cell RNAseq Analysis of lncRNAs.
  26. Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response.
  27. Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans.
  28. Experimental Validation of the Noncoding Potential for lncRNAs.
  29. Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.
  30. Detection of Circulating RNA Using Nanopore Sequencing.
  31. Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.
  32. Inhibition of dynamin-related protein 1 ameliorates the mitochondrial ultrastructure via PINK1 and Parkin in the mice model of Parkinson's disease.
  33. Quantitative analysis of mitochondrial ATP synthesis.
  34. The clinical variations and diagnostic challenges of deoxyguanosine kinase deficiency: a descriptive case series.
  35. Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-specific Enzyme.
  36. Intensive nutrition support may benefit patients with a rare mitochondrial disorder.
  37. Regulation of Skeletal Muscle Satellite Cell Differentiation by Omega-3 Polyunsaturated Fatty Acids: A Critical Review.
  38. Mitochondrial STAT5A promotes metabolic remodeling and the Warburg effect by inactivating the pyruvate dehydrogenase complex.
  39. Mechanism of Mitophagy and Its Role in Sepsis Induced Organ Dysfunction: A Review.
  40. An update on methods and approaches for interrogating mitochondrial reactive oxygen species production.
  41. Mitochondrial DNA copy number and trimethylamine levels in the blood: New insights on cardiovascular disease biomarkers.
  42. Comprehensive identification of transposable element insertions using multiple sequencing technologies.
  43. Bioinformatic Pipelines to Analyze lncRNAs RNAseq Data.
  44. Gut microbiota mediate the FGF21 adaptive stress response to chronic dietary protein-restriction in mice.
  45. Repurposing tRNAs for nonsense suppression.
  46. Mitochondria-targeted ratiometric fluorescent imaging of cysteine.
  47. DIAproteomics: A Multifunctional Data Analysis Pipeline for Data-Independent Acquisition Proteomics and Peptidomics.
  48. Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification.
  1. Dis Model Mech. 2021 Jun 01. pii: dmm048912. [Epub ahead of print]14(6):
    Mitochondrial function in development and disease.
    Marlies P Rossmann, Sonia M Dubois, Suneet Agarwal, Leonard I Zon.
      Mitochondria are organelles with vital functions in almost all eukaryotic cells. Often described as the cellular 'powerhouses' due to their essential role in aerobic oxidative phosphorylation, mitochondria perform many other essential functions beyond energy production. As signaling organelles, mitochondria communicate with the nucleus and other organelles to help maintain cellular homeostasis, allow cellular adaptation to diverse stresses, and help steer cell fate decisions during development. Mitochondria have taken center stage in the research of normal and pathological processes, including normal tissue homeostasis and metabolism, neurodegeneration, immunity and infectious diseases. The central role that mitochondria assume within cells is evidenced by the broad impact of mitochondrial diseases, caused by defects in either mitochondrial or nuclear genes encoding for mitochondrial proteins, on different organ systems. In this Review, we will provide the reader with a foundation of the mitochondrial 'hardware', the mitochondrion itself, with its specific dynamics, quality control mechanisms and cross-organelle communication, including its roles as a driver of an innate immune response, all with a focus on development, disease and aging. We will further discuss how mitochondrial DNA is inherited, how its mutation affects cell and organismal fitness, and current therapeutic approaches for mitochondrial diseases in both model organisms and humans.
    Keywords:  Mitochondrial diseases; Mitochondrial fusion and fission; Mitochondrial unfolded protein response; Mitophagy; mtDNA heteroplasmy and inheritance; mtDNA-mediated innate immune response
    DOI:  https://doi.org/10.1242/dmm.048912
  2. Brain. 2021 Jun 24. pii: awab238. [Epub ahead of print]
    Myopathic mitochondrial DNA depletion syndrome associated with biallelic variants in LIG3.
    Federica Invernizzi, Andrea Legati, Alessia Nasca, Eleonora Lamantea, Barbara Garavaglia, Mjriana Gusic, Robert Kopajtich, Holger Prokisch, Massimo Zeviani, Costanza Lamperti, Daniele Ghezzi.
      
    DOI:  https://doi.org/10.1093/brain/awab238
  3. Front Neurol. 2021 ;12 648916
    Exploiting hiPSCs in Leber's Hereditary Optic Neuropathy (LHON): Present Achievements and Future Perspectives.
    Camille Peron, Alessandra Maresca, Andrea Cavaliere, Angelo Iannielli, Vania Broccoli, Valerio Carelli, Ivano Di Meo, Valeria Tiranti.
      More than 30 years after discovering Leber's hereditary optic neuropathy (LHON) as the first maternally inherited disease associated with homoplasmic mtDNA mutations, we still struggle to achieve effective therapies. LHON is characterized by selective degeneration of retinal ganglion cells (RGCs) and is the most frequent mitochondrial disease, which leads young people to blindness, in particular males. Despite that causative mutations are present in all tissues, only a specific cell type is affected. Our deep understanding of the pathogenic mechanisms in LHON is hampered by the lack of appropriate models since investigations have been traditionally performed in non-neuronal cells. Effective in-vitro models of LHON are now emerging, casting promise to speed our understanding of pathophysiology and test therapeutic strategies to accelerate translation into clinic. We here review the potentials of these new models and their impact on the future of LHON patients.
    Keywords:  Leber's hereditary optic neuropathy; human induced pluripotent stem cells; mitochondrial disorders; organoids; retinal ganglion cells (RGC)
    DOI:  https://doi.org/10.3389/fneur.2021.648916
  4. J Biol Chem. 2021 Jun 19. pii: S0021-9258(21)00704-3. [Epub ahead of print] 100904
    Mitochondrial metabolism regulates macrophage biology.
    Yafang Wang, Na Li, Xin Zhang, Tiffany Horng.
      Mitochondria are critical for regulation of the activation, differentiation, and survival of macrophages and other immune cells. In response to various extracellular signals, such as microbial or viral infection, changes to mitochondrial metabolism and physiology could underlie the corresponding state of macrophage activation. These changes include alterations of oxidative metabolism, mitochondrial membrane potential, and tricarboxylic acid (TCA) cycling, as well as the release of mitochondrial reactive oxygen species (mtROS) and mitochondrial DNA (mtDNA) and transformation of the mitochondrial ultrastructure. Here, we provide an updated review of how changes in mitochondrial metabolism and various metabolites such as fumarate, succinate, and itaconate coordinate to guide macrophage activation to distinct cellular states, thus clarifying the vital link between mitochondria metabolism and immunity. We also discuss how in disease settings, mitochondrial dysfunction and oxidative stress contribute to dysregulation of the inflammatory response. Therefore, mitochondria are a vital source of dynamic signals that regulate macrophage biology to fine-tune immune responses.
    Keywords:  macrophage activation; macrophage biology; mitochondrial dysfunction; mitochondrial metabolism; oxidative stress
    DOI:  https://doi.org/10.1016/j.jbc.2021.100904
  5. Cell Death Dis. 2021 Jun 25. 12(7): 651
    Parp mutations protect from mitochondrial toxicity in Alzheimer's disease.
    Yizhou Yu, Giorgio Fedele, Ivana Celardo, Samantha H Y Loh, L Miguel Martins.
      Alzheimer's disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer's disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD+) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Here we analysed the metabolomic changes in flies overexpressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD+ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD+ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer's disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer's disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B are associated with a decrease in the risk and severity of Alzheimer's disease. We suggest that enhancing the availability of NAD+ by either vitamin B supplements or the inhibition of NAD+-dependent enzymes such as PARPs are potential therapies for Alzheimer's disease.
    DOI:  https://doi.org/10.1038/s41419-021-03926-y
  6. Cell Chem Biol. 2021 Jun 08. pii: S2451-9456(21)00260-9. [Epub ahead of print]
    Light-inducible deformation of mitochondria in live cells.
    Yutong Song, Peiyuan Huang, Xiaoying Liu, Zhihao Zhao, Yijin Wang, Bianxiao Cui, Liting Duan.
      Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.
    Keywords:  cryptochrome 2; light-gated hetero-dimerization; mitochondria; mitochondrial morphology; molecular motor; optical dimerizer; optogenetics; organelle mechanobiology
    DOI:  https://doi.org/10.1016/j.chembiol.2021.05.015
  7. Hum Mol Genet. 2021 Jun 24. pii: ddab156. [Epub ahead of print]
    Mitochondrial DNA disorders: From pathogenic variants to preventing transmission.
    Tiago M Bernardino Gomes, Yi Shiau Ng, Sarah J Pickett, Doug M Turnbull, Amy E Vincent.
      Mitochondrial DNA (mtDNA) disorders are recognised as one of the most common causes of inherited metabolic disorders. The mitochondrial genome occurs in multiple copies resulting in both homoplasmic and heteroplasmic pathogenic mtDNA variants. A biochemical defect arises when the pathogenic variant level reaches a threshold, which differs between variants. Moreover, variants can segregate, clonally expand, or be lost from cellular populations resulting in a dynamic and tissue-specific mosaic pattern of oxidative deficiency. MtDNA is maternally inherited but transmission patterns of heteroplasmic pathogenic variants are complex. During oogenesis, a mitochondrial bottleneck results in offspring with widely differing variant levels to their mother, whilst highly deleterious variants, such as deletions, are not transmitted. Complemented by a complex interplay between mitochondrial and nuclear genomes, these peculiar genetics produce marked phenotypic variation, posing challenges to the diagnosis and clinical management of patients. Novel therapeutic compounds and several genetic therapies are currently under investigation, but proven disease-modifying therapies remain elusive. Women who carry pathogenic mtDNA variants require bespoke genetic counselling to determine their reproductive options. Recent advances in in vitro fertilisation techniques, have greatly improved reproductive choices, but are not without their challenges. Since the first pathogenic mtDNA variants were identified over thirty years ago, there has been remarkable progress in our understanding of these diseases. However, many questions remain unanswered and future studies are required to investigate the mechanisms of disease progression and to identify new disease-specific therapeutic targets.
    DOI:  https://doi.org/10.1093/hmg/ddab156
  8. Redox Biol. 2021 Jun 08. pii: S2213-2317(21)00196-8. [Epub ahead of print]45 102037
    A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients.
    Cláudia M Deus, Susana P Pereira, Teresa Cunha-Oliveira, José Teixeira, Rui F Simões, Fernando Cagide, Sofia Benfeito, Fernanda Borges, Nuno Raimundo, Paulo J Oliveira.
      Parkinson's Disease (PD) is a neurodegenerative disorder affecting more than 10 million people worldwide. Currently, PD has no cure and no early diagnostics methods exist. Mitochondrial dysfunction is presented in the early stages of PD, and it is considered an important pathophysiology component. We have previously developed mitochondria-targeted hydroxycinnamic acid derivatives, presenting antioxidant and iron-chelating properties, and preventing oxidative stress in several biological models of disease. We have also demonstrated that skin fibroblasts from male sporadic PD patients (sPD) presented cellular and mitochondrial alterations, including increased oxidative stress, hyperpolarized and elongated mitochondria and decreased respiration and ATP levels. We also showed that forcing mitochondrial oxidative phosphorylation (OXPHOS) in sPD fibroblasts uncovers metabolic defects that were otherwise hidden. In this work, we tested the hypothesis that a lead mitochondria-targeted hydroxycinnamic acid derivative would revert the phenotype found in skin fibroblasts from sPD patients. Our results demonstrated that treating human skin fibroblasts from sPD patients with non-toxic concentrations of AntiOxCIN4 restored mitochondrial membrane potential and mitochondrial fission, decreased autophagic flux, and enhanced cellular responses to stress by improving the cellular redox state and decreasing reactive oxygen species (ROS) levels. Besides, fibroblasts from sPD patients treated with AntiOxCIN4 showed increased maximal respiration and metabolic activity, converting sPD fibroblasts physiologically more similar to their sex- and age-matched healthy controls. The positive compound effect was reinforced using a supervised machine learning model, confirming that AntiOxCIN4 treatment converted treated fibroblasts from sPD patients closer to the phenotype of control fibroblasts. Our data points out a possible mechanism of AntiOxCIN4 action contributing to a deeper understanding of how the use of mitochondria-targeted antioxidants based on a polyphenol scaffold can be used as potential drug candidates for delaying PD progression, validating the use of fibroblasts from sPD patients with more active OXPHOS as platforms for mitochondria-based drug development.
    Keywords:  Human skin fibroblasts; Metabolism; Mitochondria; Mitochondriotropic antioxidant; Sporadic Parkinson's disease
    DOI:  https://doi.org/10.1016/j.redox.2021.102037
  9. Clin Neuropathol. 2021 Jun 25.
    Mitochondrial myopathy with a unique presentation of facial and bulbar muscle weakness caused by single large scale mitochondrial DNA deletion.
    Rui Ban, Chuanqiang Pu, Fang Fang, Qiang Shi.
      
    DOI:  https://doi.org/10.5414/NP301350
  10. Lancet Neurol. 2021 Jul;pii: S1474-4422(21)00098-3. [Epub ahead of print]20(7): 573-584
    Mitochondrial disease in adults: recent advances and future promise.
    Yi Shiau Ng, Laurence A Bindoff, Gráinne S Gorman, Thomas Klopstock, Cornelia Kornblum, Michelangelo Mancuso, Robert McFarland, Carolyn M Sue, Anu Suomalainen, Robert W Taylor, David R Thorburn, Doug M Turnbull.
      Mitochondrial diseases are some of the most common inherited neurometabolic disorders, and major progress has been made in our understanding, diagnosis, and treatment of these conditions in the past 5 years. Development of national mitochondrial disease cohorts and international collaborations has changed our knowledge of the spectrum of clinical phenotypes and natural history of mitochondrial diseases. Advances in high-throughput sequencing technologies have altered the diagnostic algorithm for mitochondrial diseases by increasingly using a genetics-first approach, with more than 350 disease-causing genes identified to date. While the current management strategy for mitochondrial disease focuses on surveillance for multisystem involvement and effective symptomatic treatment, new endeavours are underway to find better treatments, including repurposing current drugs, use of novel small molecules, and gene therapies. Developments made in reproductive technology offer women the opportunity to prevent transmission of DNA-related mitochondrial disease to their children.
    DOI:  https://doi.org/10.1016/S1474-4422(21)00098-3
  11. Dev Cell. 2021 Jun 24. pii: S1534-5807(21)00481-0. [Epub ahead of print]
    Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.
    Christina G Towers, Darya K Wodetzki, Jacqueline Thorburn, Katharine R Smith, M Cecilia Caino, Andrew Thorburn.
      Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome formation caused by inactivation of ATG7 or RB1CC1/FIP200. We discovered rare surviving autophagy-deficient clones that adapted to maintain mitochondrial homeostasis after gene inactivation and identified two enhanced mechanisms affecting mitochondria including mitochondrial dynamics and mitochondrial-derived vesicles (MDVs). To further understand these mechanisms, we quantified MDVs via flow cytometry and confirmed an SNX9-mediated mechanism necessary for flux of MDVs to lysosomes. We show that the autophagy-dependent cells acquire unique dependencies on these processes, indicating that these alternate forms of mitochondrial homeostasis compensate for loss of autophagy to maintain mitochondrial health.
    Keywords:  ATG7; FIP200; SNX9; autophagy; cancer; late endosomes; mitochondria; mitochondrial dynamics; mitochondrial-derived vesicles; mitophagy
    DOI:  https://doi.org/10.1016/j.devcel.2021.06.003
  12. Alzheimers Dement. 2021 Jun 20.
    Association of mitochondrial variants and haplogroups identified by whole exome sequencing with Alzheimer's disease.
    Xiaoling Zhang, John J Farrell, Tong Tong, Junming Hu, Congcong Zhu, , Li-San Wang, Richard Mayeux, Jonathan L Haines, Margaret A Pericak-Vance, Gerard D Schellenberg, Kathryn L Lunetta, Lindsay A Farrer.
      INTRODUCTION: Findings regarding the association between mitochondrial DNA (mtDNA) variants and Alzheimer's disease (AD) are inconsistent.METHODS: We developed a pipeline for accurate assembly and variant calling in mitochondrial genomes embedded within whole exome sequences (WES) from 10,831 participants from the Alzheimer's Disease Sequencing Project (ADSP). Association of AD risk was evaluated with each mtDNA variant and variants located in 1158 nuclear genes related to mitochondrial function using the SCORE test. Gene-based tests were performed using SKAT-O.
    RESULTS: Analysis of 4220 mtDNA variants revealed study-wide significant association of AD with a rare MT-ND4L missense variant (rs28709356; minor allele frequency = 0.002; P = 7.3 × 10-5 ) as well as with MT-ND4L in a gene-based test (P = 6.71 × 10-5 ). Significant association was also observed with a MT-related nuclear gene, TAMM41, in a gene-based test (P = 2.7 × 10-5 ). The expression of TAMM41 was lower in AD cases than controls (P = .00046) or mild cognitive impairment cases (P = .03).
    DISCUSSION: Significant findings in MT-ND4L and TAMM41 provide evidence for a role of mitochondria in AD.
    Keywords:  Alzheimer's disease; genetic association; mitochondrial haplogroup; mitochondrial variant calling; whole exome sequencing
    DOI:  https://doi.org/10.1002/alz.12396
  13. Biol Rev Camb Philos Soc. 2021 Jun 21.
    The mitochondrial permeability transition pore: an evolving concept critical for cell life and death.
    Giampaolo Morciano, Natalia Naumova, Piotr Koprowski, Sara Valente, Vilma A Sardão, Yaiza Potes, Alessandro Rimessi, Mariusz R Wieckowski, Paulo J Oliveira.
      In this review, we summarize current knowledge of perhaps one of the most intriguing phenomena in cell biology: the mitochondrial permeability transition pore (mPTP). This phenomenon, which was initially observed as a sudden loss of inner mitochondrial membrane impermeability caused by excessive calcium, has been studied for almost 50 years, and still no definitive answer has been provided regarding its mechanisms. From its initial consideration as an in vitro artifact to the current notion that the mPTP is a phenomenon with physiological and pathological implications, a long road has been travelled. We here summarize the role of mitochondria in cytosolic calcium control and the evolving concepts regarding the mitochondrial permeability transition (mPT) and the mPTP. We show how the evolving mPTP models and mechanisms, which involve many proposed mitochondrial protein components, have arisen from methodological advances and more complex biological models. We describe how scientific progress and methodological advances have allowed milestone discoveries on mPTP regulation and composition and its recognition as a valid target for drug development and a critical component of mitochondrial biology.
    Keywords:  calcium; cell death; mitochondria; mitochondrial permeability transition pore
    DOI:  https://doi.org/10.1111/brv.12764
  14. Dtsch Arztebl Int. 2021 Oct 01. pii: arztebl.m2021.0251. [Epub ahead of print]
    Mitochondrial Disorders.
    Thomas Klopstock, Claudia Priglinger, Ali Yilmaz, Cornelia Kornblum, Felix Distelmaier, Holger Prokisch.
      BACKGROUND: Mitochondrial disorders are among the most common heritable diseases, with an overall lifetime risk of approximately one in 1500. Nonetheless, their diagnosis is often missed because of their extreme phenotypic and genotypic heterogeneity.METHODS: This review is based on publications retrieved by a selective literature search on the clinical features, genetics, pathogenesis, diagnosis, and treatment of mitochondrial diseases.
    RESULTS: Pathogenic defects of energy metabolism have been described to date in over 400 genes. Only a small number of these genes lie in the mitochondrial DNA; the corresponding diseases are either maternally inherited or of sporadic distribution. The remaining diseaseassociated genes are coded in nuclear DNA and cause diseases that are inherited according to Mendelian rules, mostly autosomal recessive. The most severely involved organs are generally those with the highest energy requirements, including the brain, the sensory epithelia, and the extraocular, cardiac, and skeletal musculature. Typical manifestations include epileptic seizures, stroke-like episodes, hearing loss, retinopathy, external ophthalmoparesis, exercise intolerance, and diabetes mellitus. More than two manifestations of these types should arouse suspicion of a disease of energy metabolism. The severity of mitochondrial disorders ranges from very severe disease, already evident in childhood, to relatively mild disease arising in late adulthood. The diagnosis is usually confirmed with molecular-genetic methods. Symptomatic treatment can improve patients' quality of life. The only disease-modifying treatment that has been approved to date is idebenone for the treatment of Leber hereditary optic neuropathy. Intravitreal gene therapy has also been developed for the treatment of this disease; its approval by the European Medicines Agency is pending.
    CONCLUSION: Patients with mitochondrial diseases have highly varied manifestations and can thus present to physicians in practically any branch of medicine. A correct diagnosis is the prerequisite for genetic counseling and for the initiation of personalized treatment.
    DOI:  https://doi.org/10.3238/arztebl.m2021.0251
  15. Invest Ophthalmol Vis Sci. 2021 Jun 01. 62(7): 22
    Leber's Hereditary Optic Neuropathy Arising From the Synergy Between ND1 3635G>A Mutation and Mitochondrial YARS2 Mutations.
    Xiaofen Jin, Juanjuan Zhang, Qiuzi Yi, Feilong Meng, Jialing Yu, Yanchun Ji, Jun Q Mo, Yi Tong, Pingping Jiang, Min-Xin Guan.
      Purpose: To investigate the mechanism underlying the synergic interaction between Leber's hereditary optic neuropathy (LHON)-associated ND1 and mitochondrial tyrosyl-tRNA synthetase (YARS2) mutations.Methods: Molecular dynamics simulation and differential scanning fluorimetry were used to evaluate the structure and stability of proteins. The impact of ND1 3635G>A and YARS2 p.G191V mutations on the oxidative phosphorylation machinery was evaluated using blue native gel electrophoresis and enzymatic activities assays. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analysis of effect of mutations on autophagy was undertaken via flow cytometry for autophagic flux.
    Results: Members of one Chinese family bearing both the YARS2 p.191Gly>Val and m.3635G>A mutations exhibited much higher penetrance of optic neuropathy than those pedigrees carrying only the m.3635G>A mutation. The m.3635G>A (p.Ser110Asn) mutation altered the ND1 structure and function, whereas the p.191Gly>Val mutation affected the stability of YARS2. Lymphoblastoid cell lines harboring both m.3635G>A and p.191Gly>Val mutations revealed more reductions in the levels of mitochondrion-encoding ND1 and CO2 than cells bearing only the m.3635G>A mutation. Strikingly, both m.3635G>A and p.191Gly>Val mutations exhibited decreases in the nucleus-encoding subunits of complex I and IV. These deficiencies manifested greater defects in the stability and activities of complex I and complex IV and overproduction of ROS and promoted greater autophagy in cell lines harboring both m.3635G>A and p.191Gly>Val mutations compared with cells bearing only the m.3635G>A mutation.
    Conclusions: Our findings provide new insights into the pathophysiology of LHON arising from the synergy between ND1 3635G>A mutation and mitochondrial YARS2 mutations.
    DOI:  https://doi.org/10.1167/iovs.62.7.22
  16. Cell Death Dis. 2021 Jun 19. 12(7): 632
    Mitochondrial Ca2+ oscillation induces mitophagy initiation through the PINK1-Parkin pathway.
    Zhengying Yu, Haipeng Wang, Wanyi Tang, Shaoyang Wang, Xiaoying Tian, Yujie Zhu, Hao He.
      Dysregulation of the PINK1/Parkin-mediated mitophagy is essential to Parkinson's disease. Although important progress has been made in previous researches, the biochemical reagents that induce global and significant mitochondrial damage may still hinder deeper insights into the mechanisms of mitophagy. The origin of PINK1/Parkin pathway activation in mitophagy remains elusive. In this study, we develop an optical method, ultra-precise laser stimulation (UPLaS) that delivers a precise and noninvasive stimulation onto a submicron region in a single mitochondrial tubular structure. UPLaS excites localized mitochondrial Ca2+ (mitoCa2+) oscillations with tiny perturbation to mitochondrial membrane potential (MMP) or mitochondrial reactive oxygen species. The UPLaS-induced mitoCa2+ oscillations can directly induce PINK1 accumulation and Parkin recruitment on mitochondria. The Parkin recruitment by UPLaS requires PINK1. Our results provide a precise and noninvasive technology for research on mitophagy, which stimulates target mitochondria with little damage, and reveal mitoCa2+ oscillation directly initiates the PINK1-Parkin pathway for mitophagy without MMP depolarization.
    DOI:  https://doi.org/10.1038/s41419-021-03913-3
  17. JA Clin Rep. 2021 Jun 23. 7(1): 51
    General anesthesia with remimazolam in a patient with mitochondrial encephalomyopathy: a case report.
    Yuji Suzuki, Matsuyuki Doi, Yoshiki Nakajima.
      BACKGROUND: Systemic anesthetic management of patients with mitochondrial disease requires careful preoperative preparation to administer adequate anesthesia and address potential disease-related complications. The appropriate general anesthetic agents to use in these patients remain controversial.CASE PRESENTATION: A 54-year-old woman (height, 145 cm; weight, 43 kg) diagnosed with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes underwent elective cochlear implantation. Infusions of intravenous remimazolam and remifentanil guided by patient state index monitoring were used for anesthesia induction and maintenance. Neither lactic acidosis nor prolonged muscle relaxation occurred in the perioperative period. At the end of surgery, flumazenil was administered to antagonize sedation, which rapidly resulted in consciousness.
    CONCLUSIONS: Remimazolam administration and reversal with flumazenil were successfully used for general anesthesia in a patient with mitochondrial disease.
    Keywords:  Flumazenil; Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; Mitochondrial myopathy; Remimazolam
    DOI:  https://doi.org/10.1186/s40981-021-00454-8
  18. Eur J Neurol. 2021 Jun 24.
    Occult primary white matter impairment in Leber's hereditary optic neuropathy.
    Ling Wang, Hao Ding, Bihong T Chen, Ke Fan, Qin Tian, Miaomiao Long, Meng Liang, Dapeng Shi, Chunshui Yu, Wen Qin.
      BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a disease maternally inherited from mitochondria that predominantly impairs the retinal ganglion cells and their axons. To identify whether occult brain white matter (WM) impairment is involved, a voxel-based analysis (VBA) of diffusion metrics was carried out in LHON patients with normal-appearing brain parenchyma.METHODS: Fifty-four symptomatic LHON patients (including 22 acute LHON with vision loss <= 12 months, and 32 chronic LHON) without any visible brain lesions and 36 healthy controls (HC) were enrolled in this study. VBA was applied to quantify the WM microstructural changes of LHON patients. Finally, the associations of the severity of WM impairment with disease duration and ophthalmologic deficits were assessed.
    RESULTS: Compared with the HC, the average retinal nerve fiber layer (RNFL) thickness was significantly reduced in patients with chronic LHON, while was increased in patients with acute LHON (P < 0.05, corrected). VBA identified significantly decreased fractional anisotropy widely in WM in both the acute and chronic LHON patients, including the left anterior thalamic radiation and superior longitudinal fasciculus, and bilateral corticospinal tract, dentate nuclei, inferior longitudinal fasciculus, forceps major, and optic radiation (OR) (P < 0.05, corrected). The integrity of most WM structures (except for the OR) was correlated with neither disease duration nor RNFL thickness (P > 0.05, corrected).
    CONCLUSIONS: Occult primary impairment of widespread brain white matter is present in LHON patients. The co-existing primary and secondary white matter impairment may jointly contribute to the pathological process of LHON.
    Keywords:  Leber’s hereditary optic neuropathy; diffusion tensor imaging; mitochondrial DNA; vision loss; white matter
    DOI:  https://doi.org/10.1111/ene.14995
  19. Arch Toxicol. 2021 Jun 24.
    Mechanistic perspectives on differential mitochondrial-based neuroprotective effects of several carnitine forms in Alzheimer's disease in vitro model.
    Sandra I Mota, Inês Pita, Rodolfo Águas, Slah Tagorti, Ashraf Virmani, Frederico C Pereira, A Cristina Rego.
      Mitochondrial deregulation has emerged as one of the earliest pathological events in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. Improvement of mitochondrial function in AD has been considered a relevant therapeutic approach. L-carnitine (LC), an amino acid derivative involved in the transport of long-chain fatty acids into mitochondria, was previously demonstrated to improve mitochondrial function, having beneficial effects in neurological disorders; moreover, acetyl-L-carnitine (ALC) is currently under phase 4 clinical trial for AD (ClinicalTrials.gov NCT01320527). Thus, in the present study, we investigated the impact of different forms of carnitines, namely LC, ALC and propionyl-L-carnitine (PLC) on mitochondrial toxicity induced by amyloid-beta peptide 1-42 oligomers (AβO; 1 μM) in mature rat hippocampal neurons. Our results indicate that 5 mM LC, ALC and PLC totally rescued the mitochondrial membrane potential and alleviated both the decrease in oxygen consumption rates and the increase in mitochondrial fragmentation induced by AβO. These could contribute to the prevention of neuronal death by apoptosis. Moreover, only ALC ameliorated AβO-evoked changes in mitochondrial movement by reducing the number of stationary mitochondria and promoting reversal mitochondrial movement. Data suggest that carnitines (LC, ALC and PLC) may act differentially to counteract changes in mitochondrial function and movement in neurons subjected to AβO, thus counteracting AD-related pathological phenotypes.
    Keywords:  Acetyl-L-carnitine; Alzheimer’s disease; Amyloid beta peptide oligomers; L-Carnitine; Mitochondrial dysfunction; Mitochondrial membrane potential; Neuritic mitochondrial movement; Oxygen consumption rate; Propionyl-L-carnitine
    DOI:  https://doi.org/10.1007/s00204-021-03104-1
  20. J Cell Mol Med. 2021 Jun 23.
    Advances in the mechanism and treatment of mitochondrial quality control involved in myocardial infarction.
    Chunfang Wang, Leiling Liu, Yishu Wang, Danyan Xu.
      Mitochondria are important organelles in eukaryotic cells. Normal mitochondrial homeostasis is subject to a strict mitochondrial quality control system, including the strict regulation of mitochondrial production, fission/fusion and mitophagy. The strict and accurate modulation of the mitochondrial quality control system, comprising the mitochondrial fission/fusion, mitophagy and other processes, can ameliorate the myocardial injury of myocardial ischaemia and ischaemia-reperfusion after myocardial infarction, which plays an important role in myocardial protection after myocardial infarction. Further research into the mechanism will help identify new therapeutic targets and drugs for the treatment of myocardial infarction. This article aims to summarize the recent research regarding the mitochondrial quality control system and its molecular mechanism involved in myocardial infarction, as well as the potential therapeutic targets in the future.
    Keywords:  ischemia-reperfusion; mitochondrial apoptosis; mitochondrial fission; mitochondrial fusion; mitochondrial quality control; myocardial infarction
    DOI:  https://doi.org/10.1111/jcmm.16744
  21. Mitochondrion. 2021 Jun 19. pii: S1567-7249(21)00083-0. [Epub ahead of print]
    Blood Mitochondrial DNA Copy Number: What Are We Counting?
    Martin Picard.
      There is growing scientific interest to develop scalable biological measures that capture mitochondrial (dys)function. Mitochondria have their own genome, the mitochondrial DNA (mtDNA), and it has been proposed that the number of mtDNA copies per cell (mtDNA copy number; mtDNAcn) reflects mitochondrial health. The common availability of stored DNA material or existing DNA sequencing data, especially from blood and other easy-to-collect samples, has made its quantification a popular approach in clinical and epidemiological studies. However, the interpretation of mtDNAcn is not univocal, and either a reduction or elevation in mtDNAcn can indicate dysfunction. The major determinants of blood-derived mtDNAcn are the heterogeneous cell type composition of leukocytes and platelet abundance, which can change with time of day, aging, and with disease. Hematopoiesis as a likely driver of blood mtDNAcn. Here we discuss the rationale and available methods to quantify mtDNAcn, the influence of blood cell type variations, and consider important gaps in knowledge that need to be resolved to maximize the scientific output around the investigation of blood mtDNAcn in humans.
    Keywords:  White blood cells; biomarker; count; leukocytes; mitochondrial function; mitochondrial genome; mitochondrion
    DOI:  https://doi.org/10.1016/j.mito.2021.06.010
  22. Antioxid Redox Signal. 2021 Jun 22.
    Mitochondrial NAD(P)+ transhydrogenase: from molecular features to physiology and disease.
    Annelise Francisco, Tiago Rezende Figueira, Roger Frigerio Castilho.
      SIGNIFICANCE: Proton-translocating NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), catalyzes a reversible reaction coupling the protonmotive force across the inner mitochondrial membrane and hydride (H-, a proton plus two electrons) transfer between the mitochondrial pools of NAD(H) and NADP(H). The forward NNT reaction is a source of NADPH in the mitochondrial matrix, fueling antioxidant and biosynthetic pathways with reductive potential. Despite the greater emphasis given to the net forward reaction, the reverse NNT reaction that oxidizes NADPH also occurs in physiological and pathological conditions. Recent Advances: NNT (dys)function has been linked to various metabolic pathways and disease phenotypes. Most of these findings have been based on spontaneous loss-of-function Nnt mutations found in the C57BL/6J mouse strain (NntC57BL/6J mutation) and disease-causing Nnt mutations in humans. The present review focuses on recent advances based on the mouse NntC57BL/6J mutation.CRITICAL ISSUES: Most studies associating NNT function with disease phenotypes have been based on comparisons of inbred mouse strains (with or without the NntC57BL/6J mutation), which creates uncertainties over the actual contribution of NNT in the context of other potential genetic modifiers.
    FUTURE DIRECTIONS: Future research might contribute to understanding the role of NNT in pathological conditions and elucidate how NNT regulates physiological signaling through its forward and reverse reactions. The importance of NNT in redox balance and tumor cell proliferation makes it a potential target of new therapeutic strategies for oxidative-stress-mediated diseases and cancer.
    DOI:  https://doi.org/10.1089/ars.2021.0111
  23. J Med Genet. 2021 Jun 25. pii: jmedgenet-2021-107769. [Epub ahead of print]
    Loss-of-function variants in DNM1 cause a specific form of developmental and epileptic encephalopathy only in biallelic state.
    Gökhan Yigit, Ruth Sheffer, Muhannad Daana, Yun Li, Emrah Kaygusuz, Hagar Mor-Shakad, Janine Altmüller, Peter Nürnberg, Liza Douiev, Silke Kaulfuss, Peter Burfeind, Bernd Wollnik, Knut Brockmann.
      BACKGROUND: Developmental and epileptic encephalopathies (DEEs) represent a group of severe neurological disorders characterised by an onset of refractory seizures during infancy or early childhood accompanied by psychomotor developmental delay or regression. DEEs are genetically heterogeneous with, to date, more than 80 different genetic subtypes including DEE31 caused by heterozygous missense variants in DNM1.METHODS: We performed a detailed clinical characterisation of two unrelated patients with DEE and used whole-exome sequencing to identify causative variants in these individuals. The identified variants were tested for cosegregation in the respective families.
    RESULTS: We excluded pathogenic variants in known, DEE-associated genes. We identified homozygous nonsense variants, c.97C>T; p.(Gln33*) in family 1 and c.850C>T; p.(Gln284*) in family 2, in the DNM1 gene, indicating that biallelic, loss-of-function pathogenic variants in DNM1 cause DEE.
    CONCLUSION: Our finding that homozygous, loss-of-function variants in DNM1 cause DEE expands the spectrum of pathogenic variants in DNM1. All parents who were heterozygous carriers of the identified loss-of-function variants were healthy and did not show any clinical symptoms, indicating that the type of mutation in DNM1 determines the pattern of inheritance.
    Keywords:  Pediatrics; epilepsy; genetics; nervous System Diseases
    DOI:  https://doi.org/10.1136/jmedgenet-2021-107769
  24. Hum Mol Genet. 2021 Jun 24. pii: ddab168. [Epub ahead of print]
    Coordinated pyruvate kinase activity is crucial for metabolic adaptation and cell survival during mitochondrial dysfunction.
    Xiaoshan Zhou, Flora Mikaeloff, Sophie Curbo, Qian Zhao, Raoul V Kuiper, Ákos Végvári, Ujjwal Neogi, Anna Karlsson.
      Deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported long survival of DGUOK knockout (Dguok-/-) mice despite low (<5%) mtDNA content in liver tissue. However, the molecular mechanisms enabling the extended survival remain unknown. Using transcriptomics, proteomics and metabolomics followed by in vitro assays, we aimed to identify the molecular pathways involved in the extended survival of the Dguok-/- mice. At the early stage, the serine synthesis and folate cycle were activated but declined later. Increased activity of the mitochondrial citric acid cycle (TCA cycle) and the urea cycle and degradation of branched chain amino acids were hallmarks of the extended lifespan in DGUOK deficiency. Furthermore, the increased synthesis of TCA cycle intermediates was supported by coordination of two pyruvate kinase genes, PKLR and PKM, indicating a central coordinating role of pyruvate kinases to support the long-term survival in mitochondrial dysfunction.
    DOI:  https://doi.org/10.1093/hmg/ddab168
  25. Methods Mol Biol. 2021 ;2348 71-90
    Single-Cell RNAseq Analysis of lncRNAs.
    Stefano Cagnin, Enrico Alessio, Raphael Severino Bonadio, Gabriele Sales.
      Mammalian genomes are pervasively transcribed and a small fraction of RNAs produced codify for proteins. The importance of noncoding RNAs for the maintenance of cell functions is well known (e.g., rRNAs, tRNAs), but only recently it was first demonstrated the involvement of microRNAs (miRNAs) in posttranscriptional regulation and then the activity of long noncoding RNAs (lncRNAs) in the regulation of miRNAs, DNA structure and protein function. LncRNAs have an expression more cell specific than other RNAs and basing on their subcellular localization exert different functions. In this book chapter we consider different protocols to evaluate the expression of lncRNAs at the single cell level using genome-wide approaches. We considered the skeletal muscle as example because the most abundant tissue in mammals involved in the regulation of metabolism and body movement. We firstly described how to isolate the smallest complete contractile system responsible for muscle metabolic and contractile traits (myofibers). We considered how to separate long and short RNAs to allow the sequencing of the full-length transcript using the SMART technique for the retrotranscription. Because of myofibers are multinucleated cells and because of it is better to perform single cell sequencing on fresh tissues we described the single-nucleus sequencing that can be applied to frozen tissues. The chapter concludes with a description of bioinformatics approaches to evaluate differential expression from single-cell or single-nucleus RNA sequencing.
    Keywords:  Bioinformatic; Long noncoding RNA; Myofiber; Single-nucleus RNA sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-1581-2_5
  26. EMBO J. 2021 Jun 21. e100715
    Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response.
    Guillermo López-Doménech, Jack H Howden, Christian Covill-Cooke, Corinne Morfill, Jigna V Patel, Roland Bürli, Damian Crowther, Nicol Birsa, Nicholas J Brandon, Josef T Kittler.
      Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.
    Keywords:  Parkinson’s disease; Rhot1; Rhot2; eIF2α; megamitochondria
    DOI:  https://doi.org/10.15252/embj.2018100715
  27. Sci Adv. 2021 Jun;pii: eabg3012. [Epub ahead of print]7(26):
    Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans.
    Giuseppe Calculli, Hyun Ju Lee, Koning Shen, Uyen Pham, Marija Herholz, Aleksandra Trifunovic, Andrew Dillin, David Vilchez.
      Protein aggregation causes intracellular changes in neurons, which elicit signals to modulate proteostasis in the periphery. Beyond the nervous system, a fundamental question is whether other organs also communicate their proteostasis status to distal tissues. Here, we examine whether proteostasis of the germ line influences somatic tissues. To this end, we induce aggregation of germline-specific PGL-1 protein in germline stem cells of Caenorhabditis elegans Besides altering the intracellular mitochondrial network of germline cells, PGL-1 aggregation also reduces the mitochondrial content of somatic tissues through long-range Wnt signaling pathway. This process induces the unfolded protein response of the mitochondria in the soma, promoting somatic mitochondrial fragmentation and aggregation of proteins linked with neurodegenerative diseases such as Huntington's and amyotrophic lateral sclerosis. Thus, the proteostasis status of germline stem cells coordinates mitochondrial networks and protein aggregation through the organism.
    DOI:  https://doi.org/10.1126/sciadv.abg3012
  28. Methods Mol Biol. 2021 ;2348 221-230
    Experimental Validation of the Noncoding Potential for lncRNAs.
    Emily A Dangelmaier, Ashish Lal.
      In recent years, long noncoding RNAs (lncRNAs) have been increasingly recognized as critical regulators of a broad spectrum of cellular processes. Recent advancements in proteomic technologies have uncovered that an abundance of noncoding genes, including lncRNAs, have been misannotated and in reality encode proteins. This revelation underscores the need to accurately determine the coding potential of lncRNAs prior to assessment of their functional mechanisms. Here, we detail numerous experimental techniques useful in the determination of lncRNA coding potential. Several of these methods are doubly useful in that they may also be employed in studying the function of a lncRNA, be it via an RNA, protein, or both.
    Keywords:  Coding potential; Long noncoding RNAs; Micropeptides; Translation; lncRNAs
    DOI:  https://doi.org/10.1007/978-1-0716-1581-2_15
  29. N Engl J Med. 2021 Jun 24. 384(25): 2406-2417
    Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.
    Jack J Collier, Claire Guissart, Monika Oláhová, Souphatta Sasorith, Florence Piron-Prunier, Fumi Suomi, David Zhang, Nuria Martinez-Lopez, Nicolas Leboucq, Angela Bahr, Silvia Azzarello-Burri, Selina Reich, Ludger Schöls, Tuomo M Polvikoski, Pierre Meyer, Lise Larrieu, Andrew M Schaefer, Hessa S Alsaif, Suad Alyamani, Stephan Zuchner, Inês A Barbosa, Charu Deshpande, Angela Pyle, Anita Rauch, Matthis Synofzik, Fowzan S Alkuraya, François Rivier, Mina Ryten, Robert McFarland, Agnès Delahodde, Thomas G McWilliams, Michel Koenig, Robert W Taylor.
      BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast.
    RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.
    CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).
    DOI:  https://doi.org/10.1056/NEJMoa1915722
  30. Methods Mol Biol. 2021 ;2348 273-284
    Detection of Circulating RNA Using Nanopore Sequencing.
    Jennifer Lindemann, Irene K Yan, Tushar Patel.
      RNA sequencing using nanopore sequencing is a powerful method for transcriptome analysis. The approach is appropriate for comprehensive profiling of the wide range of long noncoding RNAs. Use of nanopore-based sequencing can provide information on novel transcripts, sequence polymorphisms, and splicing variants, and thus has advantages over other gene expression profiling methods such as microarrays. Circulating extracellular long noncoding RNAs are of particular interest because of their potential use as biomarkers. Here, we describe a protocol for cDNA-PCR sequencing of circulating RNA for biomarker discovery in whole blood samples using commercially available kits and nanopore sequencing.
    Keywords:  Biofluids; Biomarker discovery; Long noncoding RNA; Nanopore sequencing; RNA sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-1581-2_19
  31. Cell Rep. 2021 Jun 22. pii: S2211-1247(21)00642-2. [Epub ahead of print]35(12): 109275
    Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.
    Agnese De Mario, Anna Tosatto, Julia Marie Hill, Janos Kriston-Vizi, Robin Ketteler, Denis Vecellio Reane, Gino Cortopassi, Gyorgy Szabadkai, Rosario Rizzuto, Cristina Mammucari.
      The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.
    Keywords:  FDA-approved drugs; MCU; amorolfine; benzethonium; high-throughput screening; mitochondrial Ca(2+) uptake; mitochondrial calcium uniporter; skeletal muscle hypertrophy; triple-negative breast cancer
    DOI:  https://doi.org/10.1016/j.celrep.2021.109275
  32. Eur J Pharmacol. 2021 Jun 17. pii: S0014-2999(21)00415-5. [Epub ahead of print]907 174262
    Inhibition of dynamin-related protein 1 ameliorates the mitochondrial ultrastructure via PINK1 and Parkin in the mice model of Parkinson's disease.
    Si-Tong Feng, Zhen-Zhen Wang, Yu-He Yuan, Xiao-Le Wang, Zhen-Yu Guo, Jing-Hong Hu, Xu Yan, Nai-Hong Chen, Yi Zhang.
      Parkinson's disease (PD) is the prevalent neurodegenerative disorder characterized by the degeneration of the nigrostriatal neurons. Dynamin-related protein 1 (Drp1) is a key regulator mediating mitochondrial fission and affecting mitophagy in neurons. It has been reported that the inhibition of Drp1 may be beneficial to PD. However, the role of Drp1 and mitophagy in PD remains elusive. Therefore, in this research, we investigated the role of Drp1 and the underlying mechanisms in the mice model of PD. We used the dynasore, a GTPase inhibitor, to inhibit the expression of Drp1. We found that inhibition of Drp1 could ameliorate the motor deficits and the expression of tyrosine hydroxylase in the mice of the PD model. But Drp1 inhibition did not affect mitochondria number and morphological parameters. Moreover, suppression of Drp1 up-regulated the mitochondrial expressions of PINK1 and Parkin while not affected the expressions of NIX and BNIP3. Conclusively, our findings suggest that the inhibition of Drp1 ameliorated the mitochondrial ultrastructure at least via regulating PINK1 and Parkin in the mice of the PD model. This study also implicates that inhibition of Drp1 might impact mitophagy and recover mitochondrial homeostasis in PD.
    Keywords:  Dynamin-related protein 1; Dynasore; Mitochondria; Mitophagy; Parkinson's disease
    DOI:  https://doi.org/10.1016/j.ejphar.2021.174262
  33. Math Biosci. 2021 Jun 16. pii: S0025-5564(21)00083-3. [Epub ahead of print] 108646
    Quantitative analysis of mitochondrial ATP synthesis.
    E Benjamin Randall, Marcus Hock, Rachel Lopez, Bahador Marzban, Collin Marshall, Daniel A Beard.
      We present a computational framework for analyzing and simulating mitochondrial ATP synthesis using basic thermodynamic and kinetic principles. The framework invokes detailed descriptions of the thermodynamic driving forces associated with the processes of the electron transport chain, mitochondrial ATP synthetase, and phosphate and adenine nucleotide transporters. Assembling models of these discrete processes into an integrated model of mitochondrial ATP synthesis, we illustrate how to analyze and simulate in vitro respirometry experiments and how models identified from in vitro experimental data effectively explain cardiac respiratory control in vivo. Computer codes for these analyses are embedded as Python scripts in a Jupyter Book to facilitate easy adoption and modification of the concepts developed here. This accessible framework may also prove useful in supporting educational applications. All source codes are available on at https://beards-lab.github.io/QAMAS_book/.
    Keywords:  Biochemical thermodynamics; Bioenergetics; Computational modeling; Metabolic pathways; Respiratory
    DOI:  https://doi.org/10.1016/j.mbs.2021.108646
  34. J Pediatr Endocrinol Metab. 2021 Jun 25.
    The clinical variations and diagnostic challenges of deoxyguanosine kinase deficiency: a descriptive case series.
    Neslihan Doğulu, Ceyda Tuna Kırsaçlıoğlu, Engin Köse, Aysel Ünlüsoy Aksu, Zarife Kuloğlu, Aydan Kansu, Fatma Tuba Eminoğlu.
      OBJECTIVES: Deoxyguanosine kinase (DGUOK) deficiency is one of the leading causes of the mitochondrial DNA-depletion syndromes (MDDS) associated with hepatocerebral involvement. Herein, we present four cases of DGUOK deficiency to emphasize the clinical variability of disease and the challenges in the diagnosis of DGUOK deficiency.CASE PRESENTATION: Hepatomegaly, hyperlactatemia, elevated alpha fetoprotein (AFP), alanine, and transaminase levels were detected in all patients, and cholestasis, coagulopathy, and hypotonia were common findings. All patients had a low birth weight, one patient underwent liver transplantation (LT). Clinical and laboratory findings of two patients and one patient suggested neonatal hemochromatosis and type 1 tyrosinemia, respectively. All patients were diagnosed with DGUOK deficiency by performing molecular genetic analysis.
    CONCLUSIONS: Mitochondrial DNA-depletion syndromes should be kept in mind in cases in which hypotonicity, lactic acidosis, and neonatal cholestasis are observed. DGUOK deficiency may present in different clinics suggesting neonatal hemochromatosis or tyrosinemia type 1.
    Keywords:  DGUOK deficiency; liver failure; neonatal cholestasis
    DOI:  https://doi.org/10.1515/jpem-2021-0108
  35. Angew Chem Int Ed Engl. 2021 Jun 19.
    Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-specific Enzyme.
    Haobin Chen, Jiangbo Yu, Jicheng Zhang, Kai Sun, Zhaoyang Ding, Yifei Jiang, Qiongzheng Hu, Changfeng Wu, Daniel T Chiu.
      Medical biosensors for real-time measurement of patient metabolite levels are needed to improve the diagnosis and management of diseases such as diabetes, phenylketonuria, and nonalcoholic fatty liver disease. Over 100 medically relevant metabolites can be quantified by measuring the stoichiometric formation of NAD(P)H in enzyme-catalyzed reactions. Here we introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and β-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use. This biosensor has broad potential utility as any metabolite that is enzymatically oxidized by NAD + or NADP + can be detected with this system.
    Keywords:  metabolite biosensor; nicotinamide adenine dinucleotide; paper assay; point of care; semiconducting polymer dots
    DOI:  https://doi.org/10.1002/anie.202106156
  36. Nutr Clin Pract. 2021 Jun 25.
    Intensive nutrition support may benefit patients with a rare mitochondrial disorder.
    Arezina Kasti, Maroulla Nikolaki, Ioannis Pyrousis, Kalliopi Synodinou, Nikolaos Oikonomopoulos, Konstantinos Triantafyllou.
      Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare, inherited, multisystemic autosomal recessive disorder caused by mutations in the nuclear TYMP gene. This syndrome is characterized by ptosis, external ophthalmoplegia, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Our case illustrates a patient diagnosed with MNGIE and cachexia who has benefited from the initiation and maintenance of parenteral nutrition. We highlight the benefits of receiving long-term supplementary home parenteral nutrition under close monitoring for patients with this neurogastrointestinal disease in order to gain weight and maintain good health.
    Keywords:  cachexia; encephalopathy; gastrointestinal disease; mitochondrial encephalomyopathies; parenteral nutrition
    DOI:  https://doi.org/10.1002/ncp.10726
  37. Front Physiol. 2021 ;12 682091
    Regulation of Skeletal Muscle Satellite Cell Differentiation by Omega-3 Polyunsaturated Fatty Acids: A Critical Review.
    Peter O Isesele, Vera C Mazurak.
      Skeletal muscle is composed of multinuclear cells called myofibres, which are formed by the fusion of myoblasts during development. The size of the muscle fiber and mass of skeletal muscle are altered in response to several pathological and physiological conditions. Skeletal muscle regeneration is primarily mediated by muscle stem cells called satellite cells (SCs). In response to injury, these SCs replenish myogenic progenitor cells to form new myofibers to repair damaged muscle. During myogenesis, activated SCs proliferate and differentiate to myoblast and then fuse with one another to form muscle fibers. A reduced number of SCs and an inability to undergo myogenesis may contribute to skeletal muscle disorders such as atrophy, cachexia, and sarcopenia. Myogenic regulatory factors (MRF) are transcription factors that regulate myogenesis and determines whether SCs will be in the quiescent, activated, committed, or differentiated state. Mitochondria oxidative phosphorylation and oxidative stress play a role in the determination of the fate of SCs. The potential activation and function of SCs are also affected by inflammation during skeletal muscle regeneration. Omega-3 polyunsaturated fatty acids (PUFAs) show promise to reduce inflammation, maintain muscle mass during aging, and increase the functional capacity of the muscle. The aim of this critical review is to highlight the role of omega-3 PUFAs on the myogenic differentiation of SCs and pathways affected during the differentiation process, including mitochondrial function and inflammation from the current body of literature.
    Keywords:  inflammation; myogenesis; omega-3; satellite cell; skeletal muscle
    DOI:  https://doi.org/10.3389/fphys.2021.682091
  38. Cell Death Dis. 2021 Jun 19. 12(7): 634
    Mitochondrial STAT5A promotes metabolic remodeling and the Warburg effect by inactivating the pyruvate dehydrogenase complex.
    Liang Zhang, Jianong Zhang, Yan Liu, Pingzhao Zhang, Ji Nie, Rui Zhao, Qin Shi, Huiru Sun, Dongyue Jiao, Yingji Chen, Xiaying Zhao, Yan Huang, Yao Li, Jian-Yuan Zhao, Wei Xu, Shi-Min Zhao, Chenji Wang.
      Signal transducer and activator 5a (STAT5A) is a classical transcription factor that plays pivotal roles in various biological processes, including tumor initiation and progression. A fraction of STAT5A is localized in the mitochondria, but the biological functions of mitochondrial STAT5A remain obscure. Here, we show that STAT5A interacts with pyruvate dehydrogenase complex (PDC), a mitochondrial gatekeeper enzyme connecting two key metabolic pathways, glycolysis and the tricarboxylic acid cycle. Mitochondrial STAT5A disrupts PDC integrity, thereby inhibiting PDC activity and remodeling cellular glycolysis and oxidative phosphorylation. Mitochondrial translocation of STAT5A is increased under hypoxic conditions. This strengthens the Warburg effect in cancer cells and promotes in vitro cell growth under hypoxia and in vivo tumor growth. Our findings indicate distinct pro-oncogenic roles of STAT5A in energy metabolism, which is different from its classical function as a transcription factor.
    DOI:  https://doi.org/10.1038/s41419-021-03908-0
  39. Front Cell Dev Biol. 2021 ;9 664896
    Mechanism of Mitophagy and Its Role in Sepsis Induced Organ Dysfunction: A Review.
    Cheng-Long Zhu, Ren-Qi Yao, Lu-Xi Li, Peng Li, Jian Xie, Jia-Feng Wang, Xiao-Ming Deng.
      Autophagy, an evolutionarily conserved process, plays an important role in maintaining cellular homeostasis under physiological and pathophysiological conditions. It is widely believed that mitochondria influence the development of disease by regulating cellular metabolism. When challenged by different stimuli, mitochondria may experience morphological disorders and functional abnormalities, leading to a selective form of autophagy-mitophagy, which can clear damaged mitochondria to promote mitochondrial quality control. Sepsis is a complex global problem with multiple organ dysfunction, often accompanied by manifold mitochondrial damage. Recent studies have shown that autophagy can regulate both innate and acquired immune processes to protect against organ dysfunction in sepsis. Sepsis-induced mitochondrial dysfunction may play a pathophysiological role in the initiation and progression of sepsis-induced organ failure. Mitophagy is reported to be beneficial for sepsis by eliminating disabled mitochondria and maintaining homeostasis to protect against organ failure. In this review, we summarize the recent findings and mechanisms of mitophagy and its involvement in septic organ dysfunction as a potential therapeutic target.
    Keywords:  autophagy; mitochondria; mitophagy; organ dysfunction; sepsis
    DOI:  https://doi.org/10.3389/fcell.2021.664896
  40. Redox Biol. 2021 Jun 16. pii: S2213-2317(21)00203-2. [Epub ahead of print]45 102044
    An update on methods and approaches for interrogating mitochondrial reactive oxygen species production.
    Ryan J Mailloux.
      The chief ROS formed by mitochondria are superoxide (O2·-) and hydrogen peroxide (H2O2). Superoxide is converted rapidly to H2O2 and therefore the latter is the chief ROS emitted by mitochondria into the cell. Once considered an unavoidable by-product of aerobic respiration, H2O2 is now regarded as a central mitokine used in mitochondrial redox signaling. However, it has been postulated that O2·- can also serve as a signal in mammalian cells. Progress in understanding the role of mitochondrial H2O2 in signaling is due to significant advances in the development of methods and technologies for its detection. Unfortunately, the development of techniques to selectively measure basal O2·- changes has been met with more significant hurdles due to its short half-life and the lack of specific probes. The development of sensitive techniques for the selective and real time measure of O2·- and H2O2 has come on two fronts: development of genetically encoded fluorescent proteins and small molecule reporters. In 2015, I published a detailed comprehensive review on the state of knowledge for mitochondrial ROS production and how it is controlled, which included an in-depth discussion of the up-to-date methods utilized for the detection of both superoxide (O2·-) and H2O2. In the article, I presented the challenges associated with utilizing these probes and their significance in advancing our collective understanding of ROS signaling. Since then, many other authors in the field of Redox Biology have published articles on the challenges and developments detecting O2·- and H2O2 in various organisms [1-3]. There has been significant advances in this state of knowledge, including the development of novel genetically encoded fluorescent H2O2 probes, several O2·- sensors, and the establishment of a toolkit of inhibitors and substrates for the interrogation of mitochondrial H2O2 production and the antioxidant defenses utilized to maintain the cellular H2O2 steady-state. Here, I provide an update on these methods and their implementation in furthering our understanding of how mitochondria serve as cell ROS stabilizing devices for H2O2 signaling.
    Keywords:  Methods for measuring ROS; Mitochondria; Peroxide detectors; Reactive oxygen species; Superoxide probes
    DOI:  https://doi.org/10.1016/j.redox.2021.102044
  41. FASEB J. 2021 Jul;35(7): e21694
    Mitochondrial DNA copy number and trimethylamine levels in the blood: New insights on cardiovascular disease biomarkers.
    Laura Bordoni, Irene Petracci, Iwona Pelikant-Malecka, Adriana Radulska, Marco Piangerelli, Joanna J Samulak, Lukasz Lewicki, Leszek Kalinowski, Rosita Gabbianelli, Robert A Olek.
      Among cardiovascular disease (CVD) biomarkers, the mitochondrial DNA copy number (mtDNAcn) is a promising candidate. A growing attention has been also dedicated to trimethylamine-N-oxide (TMAO), an oxidative derivative of the gut metabolite trimethylamine (TMA). With the aim to identify biomarkers predictive of CVD, we investigated TMA, TMAO, and mtDNAcn in a population of 389 coronary artery disease (CAD) patients and 151 healthy controls, in association with established risk factors for CVD (sex, age, hypertension, smoking, diabetes, glomerular filtration rate [GFR]) and troponin, an established marker of CAD. MtDNAcn was significantly lower in CAD patients; it correlates with GFR and TMA, but not with TMAO. A biomarker including mtDNAcn, sex, and hypertension (but neither TMA nor TMAO) emerged as a good predictor of CAD. Our findings support the mtDNAcn as a promising plastic biomarker, useful to monitor the exposure to risk factors and the efficacy of preventive interventions for a personalized CAD risk reduction.
    Keywords:  TMAO; biomarkers; cardiovascular disease; hypertension; mitochondrial DNA; sex
    DOI:  https://doi.org/10.1096/fj.202100056R
  42. Nat Commun. 2021 06 22. 12(1): 3836
    Comprehensive identification of transposable element insertions using multiple sequencing technologies.
    Chong Chu, Rebeca Borges-Monroy, Vinayak V Viswanadham, Soohyun Lee, Heng Li, Eunjung Alice Lee, Peter J Park.
      Transposable elements (TEs) help shape the structure and function of the human genome. When inserted into some locations, TEs may disrupt gene regulation and cause diseases. Here, we present xTea (x-Transposable element analyzer), a tool for identifying TE insertions in whole-genome sequencing data. Whereas existing methods are mostly designed for short-read data, xTea can be applied to both short-read and long-read data. Our analysis shows that xTea outperforms other short read-based methods for both germline and somatic TE insertion discovery. With long-read data, we created a catalogue of polymorphic insertions with full assembly and annotation of insertional sequences for various types of retroelements, including pseudogenes and endogenous retroviruses. Notably, we find that individual genomes have an average of nine groups of full-length L1s in centromeres, suggesting that centromeres and other highly repetitive regions such as telomeres are a significant yet unexplored source of active L1s. xTea is available at https://github.com/parklab/xTea .
    DOI:  https://doi.org/10.1038/s41467-021-24041-8
  43. Methods Mol Biol. 2021 ;2348 55-69
    Bioinformatic Pipelines to Analyze lncRNAs RNAseq Data.
    Luca Agnelli, Stefania Bortoluzzi, Giancarlo Pruneri.
      RNA-sequencing could be nowadays considered the gold standard to study the coding and noncoding transcriptome. The great advantage of high-throughput sequencing in the characterization and quantification of long noncoding RNA (lncRNA) resides in its capability to capture the complexity of lncRNA transcripts configuration patterns, even in the presence of several alternative isoforms, with superior accuracy and discovery power compared to other technologies such as microarrays or PCR-based methods. In this chapter, we provide a protocol for lncRNA analysis using through high-throughput sequencing, indicating the main difficulties in the annotation pipeline and showing how an accurate evaluation of the procedure can help to minimize biased observations.
    Keywords:  RNA sequencing; Sequencing data mapping; lncRNA quantification
    DOI:  https://doi.org/10.1007/978-1-0716-1581-2_4
  44. Nat Commun. 2021 06 22. 12(1): 3838
    Gut microbiota mediate the FGF21 adaptive stress response to chronic dietary protein-restriction in mice.
    Anthony Martin, Gertrude Ecklu-Mensah, Connie W Y Ha, Gustaf Hendrick, Donald K Layman, Jack Gilbert, Suzanne Devkota.
      Chronic dietary protein-restriction can create essential amino acid deficiencies and induce metabolic adaptation through the hepatic FGF21 pathway which serves to maintain host fitness during prolonged states of nutritional imbalance. Similarly, the gut microbiome undergoes metabolic adaptations when dietary nutrients are added or withdrawn. Here we confirm previous reports that dietary protein-restriction triggers the hepatic FGF21 adaptive metabolic pathway and further demonstrate that this response is mediated by the gut microbiome and can be tuned through dietary supplementation of fibers that alter the gut microbiome. In the absence of a gut microbiome, we discover that FGF21 is de-sensitized to the effect of protein-restriction. These data suggest that host-intrinsic adaptive pathways to chronic dietary protein-restriction, such as the hepatic FGF21 pathway, may in-fact be responding first to adaptive metabolic changes in the gut microbiome.
    DOI:  https://doi.org/10.1038/s41467-021-24074-z
  45. Nat Commun. 2021 06 22. 12(1): 3850
    Repurposing tRNAs for nonsense suppression.
    Suki Albers, Bertrand Beckert, Marco C Matthies, Chandra Sekhar Mandava, Raphael Schuster, Carolin Seuring, Maria Riedner, Suparna Sanyal, Andrew E Torda, Daniel N Wilson, Zoya Ignatova.
      Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.
    DOI:  https://doi.org/10.1038/s41467-021-24076-x
  46. Analyst. 2021 Jun 25.
    Mitochondria-targeted ratiometric fluorescent imaging of cysteine.
    Ya-Nan Wei, Bo Lin, Yang Shu, Jian-Hua Wang.
      As an indispensable biothiol, cysteine (Cys) plays a critical part in cellular redox homeostasis, and pathological and physiological processes. One of the main sources of reactive oxygen species (ROS) in human cells is the substrate end of the respiratory chain in the mitochondrial inner membrane. Therefore, it is valuable to develop probes targeting mitochondria to detect Cys. In this work, we designed a novel fluorescent probe, 2-(2-(6-(acryloyloxy) naphthalen-2-yl) vinyl)-3-ethylbenzothiazol-3-ium (ANET). The naphthyl benzothiazole is the fluorophore group and the acrylate moiety is the Cys response site to avoid the interference of homocysteine (Hcy) and glutathione (GSH). ANET combines multiple strengths for detecting Cys: targeting mitochondria, ratiometric fluorescence, high selectivity, and a large Stokes shift. After ANET reacted with Cys, the fluorescence signals changed from green (λem = 525 nm) to orange red (λem = 595 nm), and the detection limit was calculated to be 74 nM through a linear relationship between ratiometric fluorescence F595/F525 and Cys concentration. The imaging of Cys was confirmed in HepG2 cells.
    DOI:  https://doi.org/10.1039/d1an00758k
  47. J Proteome Res. 2021 Jun 21.
    DIAproteomics: A Multifunctional Data Analysis Pipeline for Data-Independent Acquisition Proteomics and Peptidomics.
    Leon Bichmann, Shubham Gupta, George Rosenberger, Leon Kuchenbecker, Timo Sachsenberg, Phil Ewels, Oliver Alka, Julianus Pfeuffer, Oliver Kohlbacher, Hannes Röst.
      Data-independent acquisition (DIA) is becoming a leading analysis method in biomedical mass spectrometry. The main advantages include greater reproducibility and sensitivity and a greater dynamic range compared with data-dependent acquisition (DDA). However, the data analysis is complex and often requires expert knowledge when dealing with large-scale data sets. Here we present DIAproteomics, a multifunctional, automated, high-throughput pipeline implemented in the Nextflow workflow management system that allows one to easily process proteomics and peptidomics DIA data sets on diverse compute infrastructures. The central components are well-established tools such as the OpenSwathWorkflow for the DIA spectral library search and PyProphet for the false discovery rate assessment. In addition, it provides options to generate spectral libraries from existing DDA data and to carry out the retention time and chromatogram alignment. The output includes annotated tables and diagnostic visualizations from the statistical postprocessing and computation of fold-changes across pairwise conditions, predefined in an experimental design. DIAproteomics is well documented open-source software and is available under a permissive license to the scientific community at https://www.openms.de/diaproteomics/.
    Keywords:  automation; cloud computing; data processing; data-independent acquisition; peptidomics; proteomics; spectral library generation
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00123
  48. STAR Protoc. 2021 Jun 18. 2(2): 100589
    Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification.
    Xingfa Han, Laura L Burger, David Garcia-Galiano, Suzanne M Moenter, Martin G Myers, David P Olson, Carol F Elias.
      Here, we present an in-depth protocol for extracting ribosome-bound mRNAs in low-abundance cells of hypothalamic nuclei. mRNAs are extracted from the micropunched tissue using refined translating ribosome affinity purification. Isolated RNAs can be used for sequencing or transcript quantification. This protocol enables the identification of actively translated mRNAs in varying physiological states and can be modified for use in any neuronal subpopulation labeled with a ribo-tag. We use leptin receptor-expressing neurons as an example to illustrate the protocol. For complete details on the use and execution of this protocol, please refer to Han et al. (2020).
    Keywords:  Gene Expression; Neuroscience; Protein Biochemistry
    DOI:  https://doi.org/10.1016/j.xpro.2021.100589
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