bims-meglyc Biomed News
on Metabolic disorders affecting glycosylation
Issue of 2023‒06‒18
sixteen papers selected by
Silvia Radenkovic
Frontiers in Congenital Disorders of Glycosylation Consortium
  1. Ethical challenges and opportunities in the development and approval of novel therapeutics for rare diseases.
  2. Deciphering Protein O-GalNAcylation: Method Development and Disease Implication.
  3. Applications of long-read sequencing to Mendelian genetics.
  4. Acute Encephalopathy Caused by Inherited Metabolic Diseases.
  5. Triosephosphate Isomerase Deficiency: E105D Mutation in Unrelated Patients and Review of the Literature.
  6. Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.
  7. O-GalNAc glycosylation determines intracellular trafficking of APP and Aβ production.
  8. iPSCs-Derived Neurons and Brain Organoids from Patients.
  9. Method to Generate Dorsal Forebrain Brain Organoids from Human Pluripotent Stem Cells.
  10. Maternal Inborn Errors of Metabolism Detected in Expanded Newborn Metabolic Screening.
  11. Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases.
  12. Defined Differentiation of Human Pluripotent Stem Cells to Brain Microvascular Endothelial-Like Cells for Modeling the Blood-Brain Barrier.
  13. Glycosylation in extracellular vesicles: Isolation, characterization, composition, analysis and clinical applications.
  14. The role of Zn2+ in shaping intracellular Ca2+ dynamics in the heart.
  15. Merits and challenges of iPSC-derived organoids for clinical applications.
  16. Harnessing Human Pluripotent Stem Cell-Derived Pancreatic In Vitro Models for High-Throughput Toxicity Testing and Diabetes Drug Discovery.
  1. J Med Access. 2023 Jan-Dec;7:7 27550834231177507
    Ethical challenges and opportunities in the development and approval of novel therapeutics for rare diseases.
    Djurdja Djordjevic, Andrew McFadyen, James A Anderson.
      The development of novel therapeutics for rare "orphan" diseases has brought a growing tension between the desire to accelerate access to these breakthrough therapies and the need to generate quality evidence regarding their safety and efficacy. Accelerating the pace of drug development and approval may facilitate the rapid delivery of benefits to patients and cost savings for research and development, which theoretically improves affordability of drugs for the health system. However, several ethical challenges arise with expedited approval, compassionate release of drugs, and subsequent study of drugs in "real-world" settings. In this article, we explore the changing landscape of drug approval and the ethical challenges expedited approval creates for patients, caregivers, clinicians, and institutions, and propose tangible strategies to maximize the benefits of "real-world" data acquisition while mitigating risks to patients, clinicians, and institutions.
    Keywords:  Canada; Drug approval; data collection; ethics research; rare diseases; resource allocation
    DOI:  https://doi.org/10.1177/27550834231177507
  2. ACS Omega. 2023 Jun 06. 8(22): 19223-19236
    Deciphering Protein O-GalNAcylation: Method Development and Disease Implication.
    Shuang Yue, Xiaotong Wang, Wei Ge, Jiajia Li, Chuanlai Yang, Zeyang Zhou, Peng Zhang, Xiaodong Yang, Wenjin Xiao, Shuang Yang.
      Mucin-type O-glycosylation is an important protein post-translational modification that is abundantly expressed on cell surface proteins. Protein O-glycosylation plays a variety of roles in cellular biological functions including protein structure and signal transduction to the immune response. Cell surface mucins are highly O-glycosylated and are the main substance of the mucosal barrier that protects the gastrointestinal or respiratory tract from infection by pathogens or microorganisms. Dysregulation of mucin O-glycosylation may impair mucosal protection against pathogens that can invade cells to trigger infection or immune evasion. Truncated O-glycosylation, also known as Tn antigen or O-GalNAcylation, is highly upregulated in diseases such cancer, autoimmune disorders, neurodegenerative diseases, and IgA nephropathy. Characterization of O-GalNAcylation helps decipher the role of Tn antigen in physiopathology and therapy. However, the analysis of O-glycosylation, specifically the Tn antigen, remains challenging due to the lack of reliable enrichment and identification assays compared to N-glycosylation. Here, we summarize recent advances in analytical methods for O-GalNAcylation enrichment and identification and highlight the biological role of the Tn antigen in various diseases and the clinical implications of identifying aberrant O-GalNAcylation.
    DOI:  https://doi.org/10.1021/acsomega.3c01653
  3. Genome Med. 2023 Jun 14. 15(1): 42
    Applications of long-read sequencing to Mendelian genetics.
    Francesco Kumara Mastrorosa, Danny E Miller, Evan E Eichler.
      Advances in clinical genetic testing, including the introduction of exome sequencing, have uncovered the molecular etiology for many rare and previously unsolved genetic disorders, yet more than half of individuals with a suspected genetic disorder remain unsolved after complete clinical evaluation. A precise genetic diagnosis may guide clinical treatment plans, allow families to make informed care decisions, and permit individuals to participate in N-of-1 trials; thus, there is high interest in developing new tools and techniques to increase the solve rate. Long-read sequencing (LRS) is a promising technology for both increasing the solve rate and decreasing the amount of time required to make a precise genetic diagnosis. Here, we summarize current LRS technologies, give examples of how they have been used to evaluate complex genetic variation and identify missing variants, and discuss future clinical applications of LRS. As costs continue to decrease, LRS will find additional utility in the clinical space fundamentally changing how pathological variants are discovered and eventually acting as a single-data source that can be interrogated multiple times for clinical service.
    Keywords:  Genetic variation; Long-read sequencing; Medical genetics; Mendelian disorders; Structural variation
    DOI:  https://doi.org/10.1186/s13073-023-01194-3
  4. J Clin Med. 2023 May 31. pii: 3797. [Epub ahead of print]12(11):
    Acute Encephalopathy Caused by Inherited Metabolic Diseases.
    Yohei Sugiyama, Kei Murayama.
      Acute encephalopathy is a critical medical condition that typically affects previously healthy children and young adults and often results in death or severe neurological sequelae. Inherited metabolic diseases that can cause acute encephalopathy include urea cycle disorders, amino acid metabolism disorders, organic acid metabolism disorders, fatty acid metabolism disorders, mutations in the thiamine-transporter gene, and mitochondrial diseases. Although each inherited metabolic disease is rare, its overall incidence is reported as 1 in 800-2500 patients. This narrative review presents the common inherited metabolic diseases that cause acute encephalopathy. Since diagnosing inherited metabolic diseases requires specific testing, early metabolic/metanolic screening tests are required when an inherited metabolic disease is suspected. We also describe the symptoms and history associated with suspected inherited metabolic diseases, the various tests that should be conducted in case of suspicion, and treatment according to the disease group. Recent advancements made in the understanding of some of the inherited metabolic diseases that cause acute encephalopathy are also highlighted. Acute encephalopathy due to inherited metabolic diseases can have numerous different causes, and recognition of the possibility of an inherited metabolic disease as early as possible, obtaining appropriate specimens, and proceeding with testing and treatment in parallel are crucial in the management of these diseases.
    Keywords:  THTR2 (SLC19A3) gene mutation; acute encephalopathy; amino acid metabolism disorders; enoyl-CoA hydratase short-chain 1; fatty acid metabolism disorders; homocystinuria; inherited metabolic disease; liver transplantation; mitochondrial diseases; urea cycle disorders
    DOI:  https://doi.org/10.3390/jcm12113797
  5. Mol Syndromol. 2023 Jun;14(3): 231-238
    Triosephosphate Isomerase Deficiency: E105D Mutation in Unrelated Patients and Review of the Literature.
    Arzu Selamioğlu, Meryem Karaca, Mehmet Cihan Balcı, Hüseyin Kutay Körbeyli, Aslı Durmuş, Edibe Pembegül Yıldız, Serap Karaman, Gülden Fatma Gökçay.
      Introduction: Chronic haemolytic anaemia, increased susceptibility to infections, cardiomyopathy, neurodegeneration, and death in early childhood are the clinical findings of triosephosphate isomerase (TPI) deficiency, which is an ultra-rare disorder. The clinical and laboratory findings and the outcomes of 2 patients with TPI deficiency are reported, with a review of cases reported in the literature.Case Presentation: Two unrelated patients with haemolytic anaemia and neurologic findings who were diagnosed as having TPI deficiency are presented. Neonatal onset of initial symptoms was observed in both patients, and the age at diagnosis was around 2 years. The patients had increased susceptibility to infections and respiratory failure, but cardiac symptoms were not remarkable. Screening for inborn errors of metabolism revealed a previously unreported metabolic alteration determined using tandem mass spectrometry in acylcarnitine analysis, causing elevated propionyl carnitine levels in both patients. The patients had p.E105D (c.315G>C) homozygous mutations in the TPI1 gene. Although severely disabled, both patients are alive at the ages of 7 and 9 years.
    Discussion: For better management, it is important to investigate the genetic aetiology in patients with haemolytic anaemia with or without neurologic symptoms who do not have a definitive diagnosis. The differential diagnosis of elevated propionyl carnitine levels using tandem mass spectrometry screening should also include TPI deficiency.
    Keywords:  Haemolytic anaemia; Neurodegeneration; Propionyl carnitine; Triosephosphate isomerase deficiency
    DOI:  https://doi.org/10.1159/000528192
  6. Nat Chem Biol. 2023 Jun 12.
    Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.
    Lulu Chen, Qi Zhou, Pingfeng Zhang, Wei Tan, Yingge Li, Ziwen Xu, Junfeng Ma, Gary M Kupfer, Yanxin Pei, Qibin Song, Huadong Pei.
      O-linked β-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibits decreased PRPS1 O-GlcNAcylation and activity. Together, our findings establish a direct connection among O-GlcNAc signals, de novo nucleotide synthesis and human diseases, including cancer and Arts syndrome.
    DOI:  https://doi.org/10.1038/s41589-023-01354-x
  7. J Biol Chem. 2023 Jun 09. pii: S0021-9258(23)01933-6. [Epub ahead of print] 104905
    O-GalNAc glycosylation determines intracellular trafficking of APP and Aβ production.
    Yuriko Tachida, Junko Iijima, Kazuto Takahashi, Hideaki Suzuki, Yasuhiko Kizuka, Yoshiki Yamaguchi, Katsunori Tanaka, Miyako Nakano, Daisuke Takakura, Nana Kawasaki, Yuko Saito, Hiroshi Manya, Tamao Endo, Shinobu Kitazume.
      A primary pathology of Alzheimer's disease (AD) is Aβ deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aβ precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP-knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aβ production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Αβ production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.
    Keywords:  Alzheimer’s disease (AD); Amyloid precursor protein (APP); O-glycosylation; endothelial cell; intracellular trafficking
    DOI:  https://doi.org/10.1016/j.jbc.2023.104905
  8. Handb Exp Pharmacol. 2023 Jun 13.
    iPSCs-Derived Neurons and Brain Organoids from Patients.
    Wanying Zhu, Lei Xu, Xinrui Li, Hao Hu, Shuning Lou, Yan Liu.
      Induced pluripotent stem cells (iPSCs) can be differentiated into specific neurons and brain organoids by adding induction factors and small molecules in vitro, which carry human genetic information and recapitulate the development process of human brain as well as physiological, pathological, and pharmacological characteristics. Hence, iPSC-derived neurons and organoids hold great promise for studying human brain development and related nervous system diseases in vitro, and provide a platform for drug screening. In this chapter, we summarize the development of the differentiation techniques for neurons and brain organoids from iPSCs, and their applications in studying brain disease, drug screening, and transplantation.
    Keywords:  Brain organoids; Disease modeling; Induced pluripotent stem cells (iPSCs); Neurons; Transplantation therapy
    DOI:  https://doi.org/10.1007/164_2023_657
  9. Methods Mol Biol. 2023 ;2683 169-183
    Method to Generate Dorsal Forebrain Brain Organoids from Human Pluripotent Stem Cells.
    Rebecca Sebastian, Narciso S Pavon, Yoonjae Song, Karmen T Diep, ChangHui Pak.
      Region-specific brain organoids, such as dorsal forebrain brain organoid, have become increasingly useful to model early brain development. Importantly, these organoids provide an avenue to investigate mechanisms underlying neurodevelopmental disorders, as they undergo developmental milestones resembling early neocortical formation. These milestones include the generation of neural precursors which transition into intermediate cell types and subsequently to neurons and astrocytes, as well as the fulfillment of key neuronal maturation events such as synapse formation and pruning. Here we describe how to generate free-floating dorsal forebrain brain organoids from human pluripotent stem cells (hPSCs). We also describe validation of the organoids via cryosectioning and immunostaining. Additionally, we include an optimized protocol that allows high-quality dissociation of the brain organoids to live single cells, a critical step for downstream single-cell assays.
    Keywords:  Corticogenesis; Dorsal forebrain organoids; Human embryonic stem cells (hESCs); Induced-pluripotent stem cells (iPSCs); Neurodevelopment
    DOI:  https://doi.org/10.1007/978-1-0716-3287-1_13
  10. Turk Arch Pediatr. 2023 Jun 15.
    Maternal Inborn Errors of Metabolism Detected in Expanded Newborn Metabolic Screening.
    Oğuzhan Tin, Tanyel Zübarioğlu, Mehmet Şerif Cansever, Ertuğrul Kıykım, Çiğdem Aktuğlu-Zeybek.
      OBJECTIVE: Pathologic results in expanded metabolic screening tests may be due to the medications, inappropriate sampling methods, or the maternal originated inborn errors of metabolism. The aim of this study is to identify mothers with inborn errors of metabolism through the pathologic expanded metabolic screening results of their babies.MATERIALS AND METHODS: Babies who were under 1 year of age and had a pathologic result of an expanded newborn screening for inborn errors of metabolism and their mothers were included in this retrospective single-centered study. Data of expanded metabolic screening results of both babies and their mothers were recorded. Clinical and laboratory findings relevant to suspected inborn errors of metabolism due to the pathologic screening results analysis were also noted for the mothers.
    RESULTS: Seventeen babies and their mothers were enrolled. Expanded metabolic screening results were found compatible with inborn errors of metabolism in 4 (23.5%) of 17 mothers. Two of these mothers were diagnosed with 3-methylcrotonyl-CoA carboxylase deficiency and 2 mothers were diagnosed with glutaric aciduria type 1.
    CONCLUSION: Inborn errors of metabolism can present in any period of life, and this is the first study to address the importance of metabolic screening via tandem mass spectrometry in terms of early diagnosis of inborn errors of metabolism not only in pediatric aged patients but also in adulthood in Turkey. The performance of expanded metabolic screening tests may be an important step in terms of detecting maternal inborn errors of metabolism that are not diagnosed until adulthood.
    DOI:  https://doi.org/10.5152/TurkArchPediatr.2023.23009
  11. BioDrugs. 2023 Jun 14.
    Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases.
    Lauren C Testa, Kiran Musunuru.
      Collectively, genetic disorders affect approximately 350 million individuals worldwide and are a major global health burden. Despite substantial progress in identification of new disease-causing genes, variants, and molecular etiologies, nearly all rare diseases have no targeted therapeutics that can address their underlying molecular causes. Base editing (BE) and prime editing (PE), two newly described iterations of CRISPR-Cas9 genome editing, represent potential therapeutic strategies that could be used to precisely, efficiently, permanently, and safely correct patients' pathogenic variants and ameliorate disease sequelae. Unlike "standard" CRISPR-Cas9 genome editing, these technologies do not rely on double-strand break (DSB) formation, thus improving safety by decreasing the likelihood of undesired insertions and deletions (indels) at the target site. Here, we provide an overview of BE and PE, including their structures, mechanisms, and differences from standard CRISPR-Cas9 genome editing. We describe several examples of the use of BE and PE to improve rare and common disease phenotypes in preclinical models and human patients, with an emphasis on in vivo editing efficacy, safety, and delivery method. We also discuss recently developed delivery methods for these technologies that may be used in future clinical settings.
    DOI:  https://doi.org/10.1007/s40259-023-00610-9
  12. Methods Mol Biol. 2023 ;2683 113-133
    Defined Differentiation of Human Pluripotent Stem Cells to Brain Microvascular Endothelial-Like Cells for Modeling the Blood-Brain Barrier.
    Koji L Foreman, Eric V Shusta, Sean P Palecek.
      The blood-brain barrier (BBB) comprises brain microvascular endothelial cells (BMECs) that form a high-resistance cellular interface that separates the blood compartment from the brain parenchyma. An intact BBB is pivotal to maintaining brain homeostasis but also impedes the entry of neurotherapeutics. There are limited options for human-specific BBB permeability testing, however. Human pluripotent stem cell models offer a powerful tool for dissecting components of this barrier in vitro, including understanding mechanisms of BBB function, and developing strategies to improve the permeability of molecular and cellular therapeutics targeting the brain. Here, we provide a detailed, step-by-step protocol for differentiation of human pluripotent stem cells (hPSCs) to cells exhibiting key characteristics of BMECs, including paracellular and transcellular transport resistance and transporter function that enable modeling the human BBB.
    Keywords:  Blood-brain barrier; Human pluripotent stem cells; In vitro model
    DOI:  https://doi.org/10.1007/978-1-0716-3287-1_10
  13. Biotechnol Adv. 2023 Jun 10. pii: S0734-9750(23)00103-9. [Epub ahead of print] 108196
    Glycosylation in extracellular vesicles: Isolation, characterization, composition, analysis and clinical applications.
    Veronika Vrablova, Natalia Kosutova, Anna Blsakova, Aniko Bertokova, Peter Kasak, Tomas Bertok, Jan Tkac.
      This review provides a comprehensive overview of our understanding of the role that glycans play in the formation, loading and release of extracellular vesicles (EVs). The capture of EVs (typically with a size of 100-200 nm) is described, including approaches based on glycan recognition with glycan-based analysis offering highly sensitive detection of EVs. Furthermore, detailed information is provided about the use of EV glycans and glycan processing enzymes as potential biomarkers, therapeutic targets or tools applied for regenerative medicine. The review also provides a short introduction into advanced methods for the characterization of EVs, new insights into the biomolecular corona covering EVs and bioanalytical tools available for glycan analysis.
    Keywords:  Biomolecular corona; Diagnostics; Exosomes; Extracellular vesicles; Glycans; Lectins
    DOI:  https://doi.org/10.1016/j.biotechadv.2023.108196
  14. J Gen Physiol. 2023 Jul 03. pii: e202213206. [Epub ahead of print]155(7):
    The role of Zn2+ in shaping intracellular Ca2+ dynamics in the heart.
    Amy M Dorward, Alan J Stewart, Samantha J Pitt.
      Increasing evidence suggests that Zn2+ acts as a second messenger capable of transducing extracellular stimuli into intracellular signaling events. The importance of Zn2+ as a signaling molecule in cardiovascular functioning is gaining traction. In the heart, Zn2+ plays important roles in excitation-contraction (EC) coupling, excitation-transcription coupling, and cardiac ventricular morphogenesis. Zn2+ homeostasis in cardiac tissue is tightly regulated through the action of a combination of transporters, buffers, and sensors. Zn2+ mishandling is a common feature of various cardiovascular diseases. However, the precise mechanisms controlling the intracellular distribution of Zn2+ and its variations during normal cardiac function and during pathological conditions are not fully understood. In this review, we consider the major pathways by which the concentration of intracellular Zn2+ is regulated in the heart, the role of Zn2+ in EC coupling, and discuss how Zn2+ dyshomeostasis resulting from altered expression levels and efficacy of Zn2+ regulatory proteins are key drivers in the progression of cardiac dysfunction.
    DOI:  https://doi.org/10.1085/jgp.202213206
  15. Front Cell Dev Biol. 2023 ;11 1188905
    Merits and challenges of iPSC-derived organoids for clinical applications.
    Ziran Xu, Jiaxu Yang, Xianyi Xin, Chengrun Liu, Lisha Li, Xianglin Mei, Meiying Li.
      Induced pluripotent stem cells (iPSCs) have entered an unprecedented state of development since they were first generated. They have played a critical role in disease modeling, drug discovery, and cell replacement therapy, and have contributed to the evolution of disciplines such as cell biology, pathophysiology of diseases, and regenerative medicine. Organoids, the stem cell-derived 3D culture systems that mimic the structure and function of organs in vitro, have been widely used in developmental research, disease modeling, and drug screening. Recent advances in combining iPSCs with 3D organoids are facilitating further applications of iPSCs in disease research. Organoids derived from embryonic stem cells, iPSCs, and multi-tissue stem/progenitor cells can replicate the processes of developmental differentiation, homeostatic self-renewal, and regeneration due to tissue damage, offering the potential to unravel the regulatory mechanisms of development and regeneration, and elucidate the pathophysiological processes involved in disease mechanisms. Herein, we have summarized the latest research on the production scheme of organ-specific iPSC-derived organoids, the contribution of these organoids in the treatment of various organ-related diseases, in particular their contribution to COVID-19 treatment, and have discussed the unresolved challenges and shortcomings of these models.
    Keywords:  3D; COVID-19; IPSC; disease model; organoid
    DOI:  https://doi.org/10.3389/fcell.2023.1188905
  16. Handb Exp Pharmacol. 2023 Jun 13.
    Harnessing Human Pluripotent Stem Cell-Derived Pancreatic In Vitro Models for High-Throughput Toxicity Testing and Diabetes Drug Discovery.
    Carmen Ching, Elhadi Iich, Adrian Kee Keong Teo.
      The long-standing goals in diabetes research are to improve β-cell survival, functionality and increase β-cell mass. Current strategies to manage diabetes progression are still not ideal for sustained maintenance of normoglycemia, thereby increasing demand for the development of novel drugs. Available pancreatic cell lines, cadaveric islets, and their culture methods and formats, either 2D or 3D, allow for multiple avenues of experimental design to address diverse aims in the research setting. More specifically, these pancreatic cells have been employed in toxicity testing, diabetes drug screens, and with careful curation, can be optimized for use in efficient high-throughput screenings (HTS). This has since spearheaded the understanding of disease progression and related mechanisms, as well as the discovery of potential drug candidates which could be the cornerstone for diabetes treatment. This book chapter will touch on the pros and cons of the most widely used pancreatic cells, including the more recent human pluripotent stem cell-derived pancreatic cells, and HTS strategies (cell models, design, readouts) that can be used for the purpose of toxicity testing and diabetes drug discovery.
    Keywords:  Diabetes; Drug screening; High throughput; Human; Islet; Pancreas; Stem cells; Toxicity; iPS; β-cell
    DOI:  https://doi.org/10.1007/164_2023_655
This page is being maintained by Thomas Krichel. It was last updated on 2023‒06‒19 at 13:05:55.