XBP1s-EDEM2 prevents the onset and development of HFpEF by ameliorating cardiac lipotoxicity

BACKGROUND: Morbidity and mortality of heart failure with preserved ejection fraction (HFpEF) is increased in metabolic disorders. However, options for preventing and treating these prevalent outcomes are limited. Intramyocardial lipotoxicity contributes to cardiac dysfunction. Here, we investigate the mechanisms underlying endoplasmic reticulum degradation enhancing EDEM2 (endoplasmic reticulum degradation-enhancing alpha-mannosidase-like protein 2) regulation of cardiac lipid homeostasis and assess strategies that inhibit the incidence and progression of HFpEF. METHODS: Metabolic stress was induced in C57BL/6 male mice using a high-fat diet and Nω-nitro-l-arginine methyl ester. The recombinant adeno-associated virus 9 delivery system was used for loss- and gain-of-function studies. Palmitic acid and oleic acid stimulation of rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes imitated a condition of high lipids in vitro. Molecular mechanisms were investigated via RNA sequencing, mass spectrometry proteomics, lipidomic analyses, transmission electron microscopy, histology, and luciferase reporter assays. RESULTS: In the human heart, we first detected lipid overload accompanied by a reduction of XBP1 (X-box binding protein 1) under metabolic stress. Thereafter, a decrease in EDEM2 was confirmed in human and mouse HFpEF hearts. Given that the spliced form of XBP1 (XBP1s) is a transcription factor, EDEM2 was identified as its new target in cardiomyocytes. EDEM2 knockdown mice manifested lipid droplet accumulation and higher levels of triglycerides and diglycerides in the myocardium, aggravating oxidative stress, hypertrophy, and the onset and progression of HFpEF under metabolic stress. XBP1s ablation mice displayed a similar myocardial lipid disturbance and cardiac phenotypes, which were reversed by EDEM2 overexpression. Mechanistically, the findings obtained from rat cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes demonstrated that, in the presence of EDEM2, SEC23A mediated intracellular translocation of ATGL (adipose triglyceride lipase) under fatty acid stimulation, inhibiting ATGL degradation and excessive intracellular lipid droplets. Furthermore, the functional studies supported that EDEM2 prevention of lipid overload occurred in an ATGL-dependent manner. Therapeutically, cardiac XBP1s or EDEM2 restoration mitigated lipid deposition and preserved lipid profiles in the myocardium, thus preventing the development of HFpEF. CONCLUSIONS: We demonstrate a cardioprotective mechanism regulating myocardial lipid homeostasis. The findings provide a promising therapeutic target to prevent and treat HfpEF, a condition with limited treatment options

Serially quantifying TERT rearrangement breakpoints in circulating tumor DNA enables minimal residual disease monitoring in patients with neuroblastoma

Telomerase is reactivated by genomic TERT rearrangements in ~30% of diagnosed high-risk neuroblastomas. Dismal patient prognosis results if the RAS/MAPK/ALK signaling transduction network also harbors mutations. We present a liquid biopsy-based monitoring strategy for this particularly vulnerable pediatric patient subgroup, for whom real-time molecular diagnostic tools are limited to date. Droplet digital PCR assays quantifying patient-individualized TERT rearrangement breakpoint copies, ALK copy numbers and allelic ALK p.R1275Q mutation frequencies were applied to longitudinally collected liquid biopsies (peripheral blood and bone marrow plasma, n=44 biosamples), the mononuclear cell fraction from bone marrow and matched tumor samples. Marker detection was compared with current gold standard diagnostics. Reanalysis of whole-genome and targeted panel sequencing data from 169 patients identified 64 TERT-rearranged neuroblastoma samples collected at initial and/or relapse diagnosis from 55 patients (254 total TERT rearrangement events). Detection and quantification of unique TERT rearrangement breakpoints in as little as 1ng of total cell-free DNA in peripheral blood plasma improved therapy response assessment and early relapse detection in individual patients. Proof-of-concept is provided for minimal residual disease detection in the bone marrow niche, from which relapses frequently arise, by analyzing unique TERT rearrangement breakpoints in bone marrow plasma-derived cell-free DNA. TERT rearrangement breakpoints, as a single marker or combined with mutations in the RAS/MAPK/ALK signaling transduction network, can serve as robust and highly sensitive biomarkers for disease activity and spatially and temporally resolve disease better than current gold standard diagnostics in individual patients with TERT-driven neuroblastoma

An integrated landscape of mRNA and protein isoforms

Alternative splicing and proteolytic processing expand the functional diversity of the human proteome by generating distinct protein isoforms from a single gene. However, the extent to which transcript isoforms give rise to distinct protein products remains unclear, in part due to technical limitations in proteomic workflows. Here, we combine full-length mRNA sequencing with SDS-PAGE-based protein fractionation and quantitative mass spectrometry to construct an integrated landscape of mRNA and protein isoforms in human RPE-1 cells. To overcome the inherent ambiguity of bottom-up proteomics in isoform detection, we developed IsoFrac, a computational pipeline that resolves protein isoforms based on their migration profiles across gel fractions. This approach enabled the identification of ~45,000 full-length transcripts, ~32,000 ORFs, and ~16,000 distinct protein isoforms. Comparative analyses revealed widespread translation of alternative transcripts and uncovered proteolytic processing as a major, underappreciated source of proteome complexity. Our results establish a scalable framework for isoform-resolved proteogenomics and provide a reference resource for studying the molecular diversity encoded by the human genome

Herpesviruses mimic zygotic genome activation to promote viral replication

Zygotic genome activation (ZGA) is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In humans, ZGA is induced by DUX4, a pioneer factor that drives expression of downstream germline-specific genes and retroelements. Here we show that herpesviruses from all subfamilies, papillomaviruses and Merkel cell polyomavirus actively induce DUX4 expression to promote viral transcription and replication. Analysis of single-cell sequencing data sets from patients shows that viral DUX4 activation is of relevance in vivo. Herpes-simplex virus 1 (HSV-1) immediate early proteins directly induce expression of DUX4 and its target genes, which mimics zygotic genome activation. Upon HSV-1 infection, DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for infection, since genetic depletion by CRISPR/Cas9 as well as degradation of DUX4 by nanobody constructs abrogates HSV-1 replication. Our results show that DNA viruses including herpesviruses mimic an embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression

Outer retina micro-inflammation is driven by T cell responses prior to retinal degeneration in early age-related macular degeneration

INTRODUCTION: Age-related macular degeneration (AMD) is a leading cause of blindness with limited treatment options. Dysfunction of the retinal pigment epithelium (RPE) is a unifying salient feature of the pathology and a primary end-point damage leading to complications such as geographic atrophy (GA), which represents the most common end-stage of AMD. METHODS: Human and murine ocular tissues were used for histological examinations. Furthermore, flow cytometry and gene expression analysis were used on ocular and splenic tissues of Cx3cr1(GFP/GFP) and C57BL/6J mice at 8 and 12 months of age to characterize the dynamics of local and systemic T cell populations. RESULTS: We show the presence of memory T cells such as CD45RO(+) cells in the choroid and retina of patients with AMD with a peak of abundance in early stages of AMD. As further evidence for the contribution of the adaptive immune system to GA we identified an increased frequency of CD44(+) CD69(+) KLRG1(+) T cells and para-inflammation of the retina in a mouse model that mimics features of GA. Importantly, the activation of T cells found at early AMD-like stages prior to degeneration possessed long-lasting cytotoxic properties and adopted typical features of senescent immune cells. T cells were intimately associated with the RPE, suggesting transmigration and participating in local micro-inflammation. DISCUSSION: Our data support that activation and accumulation of memory T cells can be considered as a hallmark of early AMD, and that adaptive immunosenescence likely to contribute to the chronic inflammation associated with RPE damage and the progression to large lesions as seen in GA

Protocol of the follow-up of patients with transthyretin amyloid cardiomyopathy by multimodality imaging (FAITH) study: a prospective observational study in patients with ATTR-CM undergoing treatment with tafamidis

INTRODUCTION: This prospective observational study of patients with transthyretin amyloid cardiomyopathy (ATTR-CM) undergoing treatment with tafamidis aims at identifying quantitative image markers and comparing imaging modalities regarding the follow-up and prognostication of these patients, with the goal of providing a multiparametric score to predict treatment response. METHODS AND ANALYSIS: Patients with a board-approved decision to receive tafamidis will undergo, in addition to standard of care, baseline and follow-up cardiovascular magnetic resonance (CMR) scans at 9 and 18 months. In total, the study plans to recruit and scan 60 patients. A blinded read will take place in a CMR research core laboratory. The final statistical analysis will be based on developing a multiparametric score for the prediction of treatment response. The study will be managed through the Amyloidosis Center Charité Berlin, a clinical unit formed from the three clinical campus sites of the Charité in Berlin, using the Berlin Research Network for CMR. ETHICS AND DISSEMINATION: The study was approved by the Charité-Universitätsmedizin Berlin ethics committee EA1/262/23. The results of the study will be disseminated through international peer-reviewed publications and congress presentations. TRIAL REGISTRATION NUMBER: Approved WHO primary register: German Clinical Trials Register: https://www.drks.de/DRKS00033884. WHO International Clinical Registry Platform: https://trialsearch.who.int/?TrialID=DRKS00033884. Recruitment started on 1 July 2024

Identification and characterization of highly potent and isoenzyme-selective inhibitors of deiodinase type I via a nonradioactive high-throughput screening method

OBJECTIVE: Deiodinase type I (DIO1) is crucial in maintaining thyroid hormone (TH) balance. It converts the prohormone thyroxine (T4) to the active triiodothyronine (T3) and degrades T3 to inactive 3,3'-diiodothyronine (3,3'-T2). It also acts on reverse T3 (rT3) and sulfated TH metabolites, thus contributing to TH elimination. Upregulation of DIO1 is linked to hyperthyroid conditions such as Graves' disease and autonomous thyroid adenoma, making it a promising target for pharmacological intervention. The adverse side effects of the antithyroid drug propylthiouracil (PTU), used in clinics to treat hyperthyroidism due to its thyroid peroxidase- and DIO1-blocking action, highlight the need for novel and potent DIO1-selective inhibitors. METHODS: Using a semiautomatic high-throughput screening (HTS) assay based on the Sandell-Kolthoff (SK) reaction in 384-well plates, we screened 69,344 low-molecular-weight compounds for DIO1-inhibitory effects. Shortlisted hits underwent detailed manual characterization, where we evaluated the potency and isoenzyme specificity by assessing their DIO-inhibitory effects on enzyme preparations from all three DIO isoenzymes, over a wide concentration range (5 nM-20 µM). To evaluate the DIO1 inhibitory effects in intact cells, we applied a novel protocol based on the SK reaction to cell culture supernatants and assessed the intracellular deiodinase activity in DIO1 overexpressing HEK293 cells. RESULTS: The robust HTS assay flagged 436 (<1%) of the screened compounds as hits, also including known DIO1 inhibitors such as PTU and genistein. Based on a validation screen of 298 compounds, we prioritized 26 compounds to comprehensively characterize their DIO1-selective inhibition. We identified 15 DIO1-selective compounds (IC50 < 1 µM), more potent than the bonafide DIO1-selective inhibitor PTU. Additionally, 8 of the 13 tested compounds were found capable of inhibiting DIO1 in intact cells. CONCLUSIONS: With a successful SK-reaction-based HTS application, we identified novel, potent, and selective inhibitors of DIO1 with nanomolar IC50 values. Furthermore, we successfully showed that some of these compounds were also capable of inhibiting intracellular DIO1 in intact cells. These novel compounds hold immense potential in studying TH modulation, deciphering DIO1 enzyme structure, and developing structure-activity relationships. Furthermore, our novel inhibitors act as lead compounds in developing strategies to combat hyperthyroidism

Selection of therapeutically effective T-cell receptors from the diverse tumor-bearing repertoire

BACKGROUND: The development of T-cell receptor (TCR)-based T-cell therapies is hampered by the difficulties in identifying therapeutically effective tumor-specific TCRs from the natural repertoire of a patient's cancer-specific T cells. METHODS: Here, we mimic experimentally near-patient conditions to analyze the T-cell repertoire in euthymic tumor-bearing mice responding to the H-2K(b)-presented neoantigen p68(S551F) (mp68). We temporarily separated the time point of mp68 expression from that of cancer cell transplantation to exclude the influence of injection-induced inflammation on T-cell priming. Thus, the mp68-specific T-cell response could only develop after the acute inflammatory phase had subsided.Here, we mimic experimentally near-patient conditions to analyze the T-cell repertoire in euthymic tumor-bearing mice responding to the H-2K(b)-presented neoantigen p68(S551F) (mp68). We temporarily separated the time point of mp68 expression from that of cancer cell transplantation to exclude the influence of injection-induced inflammation on T-cell priming. Thus, the mp68-specific T-cell response could only develop after the acute inflammatory phase had subsided. RESULTS: We found that mp68-specific TCRs isolated from either tumor-infiltrating T cells or spleens of mice immunized with mp68-expressing cancer cells are diverse and not inherently therapeutic when introduced into peripheral T cells and used for adoptive therapy of established tumors. While measuring short-term T-cell responses in vitro was unreliable for some TCRs in predicting their therapeutic failure, assessing the persistence of cancer cell destruction by TCR-modified T cells in long-term cultures accurately predicted therapeutic outcomes. A tumor-derived TCR with optimal function was also correctly identified with this approach when analyzing human TCRs that recognize the HLA-A2-presented neoantigen CDK4(R24L). CONCLUSIONS: We show that a neoantigen-directed T-cell response in tumor-bearing hosts comprises a diverse repertoire. Infiltration and expansion of certain T-cell clonotypes in the tumor do not necessarily correlate with therapeutic efficacy of their TCRs in adoptive therapy. We propose that analysis of persistent rather than immediate responses of TCR-modified T cells in vitro serves as a reliable parameter to identify TCRs that are therapeutically effective in vivo

Dental and oral health assessments in the German National Cohort (NAKO)

BACKGROUND: Despite considerable improvements in oral health in recent decades, caries and periodontitis are still widespread, ranking among the most prevalent diseases worldwide and requiring future research. The German National Cohort (NAKO Gesundheitsstudie, NAKO) is a large-scaled, multidisciplinary, nationwide, multi-centre, population-based, prospective cohort study with oral examinations that aims to provide a resource to study risk factors for major diseases. The aim of the present article is to provide the methodological background, to report on the data quality, and to present initial results of the oral examinations. METHODS: During baseline examinations (2014-2019), a total of 205,184 persons aged 19-74 years has been examined in 18 study centres, including, among others, a dental interview, stimulated saliva sampling, and recording of the numbers of present teeth and prostheses (standard Level 1 program). As part of the Level 2 program that was offered to 20% randomly selected participants, each study centre selected one of three modules, one of them being the Level 2 oral examination. This extended program was carried out in a subgroup of 20,828 participants, including collection of detailed information on the dental and prosthetic status as well as on periodontal, cariological and functional aspects. To ensure reliability and reproducibility, study nurses were trained and calibrated by dental experts. In addition, a reliability study was conducted among 794 Level 1 and 359 Level 2 participants, reporting intra class correlation and kappa coefficients. RESULTS: Intra class correlation and kappa coefficients for observer agreement and reliability were consistently above 0.7, indicating good to excellent reliability of all dental measurements. For example, intra class correlation was 0.937 for the number of present teeth (Level 1), 0.740 for mean probing depth (PD) and 0.797 for active mouth opening. An initial inspection of the data showed that the median number of present teeth was 27, of which on average 6.9 teeth were healthy and caries-free. Average mean PD was 1.92 mm. An orthodontic treatment was reported by 35.5% of participants. DISCUSSION: Overall, the dental study protocol was feasible and successfully integrated into the NAKO's overall assessment program. However, rigorous support of the study centres by dental professionals was required to ensure high quality data. In summary, high-quality data collection within the NAKO pave the way for future investigation of potential risk factors for oral diseases and links between oral and systemic diseases and conditions