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Unlocking antigen flexibility with widened patient access: CD3FR in off-the-shelf CAR T cells
No full textMagnetic resonance cartography of renal tubule volume fraction during diuretic intervention
Get PDFAIM: The renal tubular volume fraction (TVF) fluctuates under physiological conditions, and is altered in several renal diseases. Tools that enable noninvasive assessment of TVF are currently lacking. Magnetic Resonance (MR) TVF cartography is a novel approach for unraveling renal (patho-)physiology. Here, we employ MR-TVF cartography to monitor changes in response to the diuretic furosemide, and examine its role for the interpretation of renal oxygenation assessed by mapping the MRI relaxation time T(2)*. We hypothesize that furosemide increases TVF. METHODS: In anesthetized rats (n = 7) the MRI relaxation times T(2), T(2)*, T(2)' and kidney size were obtained before/following an i.v. bolus of furosemide using a 9.4 Tesla MRI scanner. Spectral analysis of the T(2) signal decay was performed to estimate the number of T(2) components in renal tissue. TVF cartographies were calculated using voxel-wise bi-exponential fit of the T(2) decay. Near Infrared Spectroscopy (NIRS, n = 9) was used to assess the total hemoglobin concentration (HbT) as a surrogate of renal blood volume. RESULTS: Furosemide induced changes in renal MRI and NIRS parameters relative to baseline: TVF(CORTEX) = 31.1%, TVF(OUTER_MEDULLA) = 30.7%, T(2_CORTEX) = 13.0% and T2(_OUTER_MEDULLA) = 20.6%. HbT(CORTEX) was reduced by 2.7%. HbT(MEDULLA) declined by 8.6%. Kidney size showed a modest increase of 2.9%. T(2)*(OUTER_MEDULLA) and T(2)´(OUTER_MEDULLA) rose by 20.5% and 20.2%. T(2)*(CORTEX) and T(2)´(CORTEX) remained unchanged. T(2)* and TVF were strongly correlated in the outer medulla and moderately in the cortex. CONCLUSION: MR-TVF cartography is highly relevant for elucidating mechanisms of renal (patho-)physiology, including the role of renal oxygenation assessed by MRI mapping of renal T(2)*
SLAM-Seq of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
No full textUPF1 is a multi-domain RNA helicase that constantly monitors the transcriptome by non-specifically binding to mRNAs, dissociating from non-target transcripts, and initiating degradation on selected target RNAs via multiple proposed pathways such as nonsense-mediated decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. The UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). Using SLAM-Seq and this cell line, we wanted to explore the time-resolved RNA stability changes upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0h, 12h and 24h) and the cells were labeled with 200 µM 4-thiouridine (4SU) the last 2 hours before harvesting. As controls, the parental cell line (with AtAFB2-mCherry in the AAVS1 locus) or unlabeled cells were used
Rapid UPF1 depletion illuminates the temporal dynamics of the NMD-regulated human transcriptome
No full textUPF1 is a multi-domain RNA helicase that constantly monitors the transcriptome by non-specifically binding to mRNAs, dissociating from non-target transcripts, and initiating degradation on selected target RNAs via multiple proposed pathways such as nonsense-mediated decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. The UPF1 gene is essential in cultured human cells and previous studies relied mostly on RNA interference to downregulate UPF1. Here we established an auxin-inducible UPF1 degron system in the human colorectal adenocarcinoma cell line HCT116 by first inserting the auxin receptor F-box protein-encoding AtAFB2-mCherry in the AAVS1 locus, followed by tagging UPF1 at the N-terminus with an V5-AID-tag (AID = miniIAA7 = AtIAA7 amino acids 37–104). With this cell line we wanted to explore the time-resolved proteomic changes upon rapid depletion of UPF1. To this end, depletion of UPF1 was induced with 500 µM indole-3-acetic acid (IAA) for various time periods (0-24h). As controls, the cells were treated for the same time with DMSO
A noncanonical role of glycolytic metabolites controlling the timing of mouse embryo segmentation
Get PDFStudies on the impact of metabolism on cell fate decisions are seeing a renaissance. However, a key challenge remains to distinguish signaling functions of metabolism from its canonical bioenergetic and biosynthetic roles, which underlie cellular homeostasis. Here, we tackled this challenge using mouse embryonic axis segmentation as an experimental model. First, we found that energetically subminimal amounts of glucose can support ongoing segmentation clock activity, providing evidence that glycolysis exerts a signaling function. Using a dynamical systems approach based on entrainment, we identified fructose 1,6-bisphosphate (FBP) as the potential signaling metabolite. Functionally, we demonstrated that glycolytic flux/FBP control the segmentation clock period and Wnt signaling in an anticorrelated manner. Critically, we showed that the slow segmentation clock phenotype caused by elevated glycolysis is mediated by Wnt signaling rather than cellular bioenergetic and biosynthetic state. Combined, our results demonstrate a modular organization of metabolic functions, revealing a signaling module of glycolysis that can be decoupled from its canonical metabolic functions
ERV3-MLT1 provides cis-regulatory elements for human placental functioning and are commonly dysregulated in human-specific preeclampsia
No full textBACKGROUND: Owing to their transcription factor binding sites, endogenous retroviruses (ERVs) can act as cis-regulatory-elements (CREs). By invading genomes in waves, ERVs offer a substrate for lineage-specific adaptations but also, when dysregulated, for lineage-specific disorders. Their role as CREs in rapid placental evolution, and in the human-specific placental disorder preeclampsia, may thus provide a paradigmatic exemplar. Here then we systematically identify ERV-derived CREs controlling human placental gene expression with dysregulation in preeclampsia.
RESULTS: We identify 87 ERV-derived CREs located upstream of genes expressed in the placenta. A subset of nine, all belonging to the ERV3-MLT1/2 families and dating to the mouse–human common ancestor, are consistently dysregulated in trophoblasts from preeclampsia samples. Of the nine ERV3-MLT1-linked genes dysregulated in preeclampsia, five are novel candidates, while four were previously associated with preeclampsia, though their ERV-based regulation was not recognized. Focusing on EPS8L1, we predict enhancer activity of upstream MLT1(G1) and validate using reporter assay and genome editing. This vertebrate-specific gene is expressed in progenitor cytotrophoblasts and syncytiotrophoblasts and is overexpressed in preeclampsia, correlating with preeclampsia biomarkers and is not elevated in related pregnancy disorders. A soluble form of EPS8L1 is detectable in maternal plasma as early as between 24 weeks of gestation. EPS8L1 knockout in trophoblast in vitro is lethal, and its overexpression alters trophoblast behaviors characteristic of preeclampsia.
CONCLUSIONS: We conclude that ERV3-MLT1functions as a trophoblast-specific CRE for several human genes and may be dysregulated in preeclampsia. As EPS8L1 has a form in maternal circulation, it may have utility in diagnostics
Activated CaMKIIδ translocates to the RyR nanodomain in cardiomyocytes
Get PDFAIMS: The heartbeat is triggered by the coordinated release of Ca2+ from the ryanodine receptor type-2 (RyR) in cardiomyocytes. Phosphorylation of RyR by Ca2+/calmodulin-dependent kinase IIδ (CaMKIIδ) fine-tunes this process in health, while hyperphosphorylation causes excessive, pathological Ca2+ release. We investigated how CaMKIIδ is spatially recruited and anchored to RyRs to achieve this functional regulation. METHODS AND RESULTS: We employed confocal and dSTORM microscopy to investigate the macro- and nanoscale distribution of CaMKIIδ across cardiomyocytes, respectively. We linked positional rearrangement of the kinase during β-adrenergic stimulation (isoproterenol, Iso) to alterations in RyR phosphorylation and function (Ca2+ sparks), and the requirement of the CaMKIIδ anchoring protein AKAP18δ by knockdown/knockout. Confocal microscopy revealed that macroscale CaMKIIδ localization was not markedly altered during Iso-treatment, although a narrowing of its distribution around the Z-lines occurred, where the RyR reside. Higher resolution dSTORM imaging confirmed that local mobilization of CaMKIIδ by Iso decreased the distance from Z-lines and RyRs to the nearest CaMKIIδ by 28 and 12%, respectively. Functionally, kinase translocation into the RyR nanodomain was accompanied by increased channel phosphorylation and Ca2+ spark frequency. These actions were dependent on CaMKIIδ activity, since kinase translocation, RyR phosphorylation, and activation were all mimicked by the upstream activator of CaMKIIδ (8-CPT) and prevented by direct CaMKIIδ inhibitors (AIP, N1 peptide). A critical role of AKAP18δ in this mechanism was supported by immunoprecipitation experiments, which showed greater kinase binding to AKAP18δ during Iso-stimulation. Furthermore, loss of AKAP18δ by viral-mediated AKAP18δ knockdown or knockout prevented CaMKIIδ translocation to Z-lines. Microtubular disruption also blocked CaMKIIδ translocation. CONCLUSION: Collectively, our results indicate that nanoscale movement of CaMKIIδ is closely associated with RyR activation following β-adrenergic stimulation. This translocation depends on an intact microtubular network and kinase binding to AKAP18δ
Unravelling drug resistant proteotypes through phenotype-resolved proteomics of single-cell derived colonies
Get PDFDrug resistance in cancer therapy continues to significantly contribute to treatment failure and disease progression, and is linked to intratumoral heterogeneity. Mass spectrometry (MS)-based single-cell proteomics (SCP) provides a unique opportunity to uncover the mechanisms underlying drug-resistant phenotypes; however, current methods lack clonal resolution and are often confounded by cell cycle and cell size differences. Here, we introduce PhenoSCoP, a microscopy-guided discovery proteomics concept for mapping clonal proteomic heterogeneity. By distinguishing between transient and long-lived protein level changes, our approach uncovered hereditary and clone-specific programs associated with chemotherapeutic responses in head and neck squamous cell carcinoma (HNSCC) cells. Combined with fluorescence barcoding and drug treatment assays, we identified pre-existing proteotypes that strongly dominated drug-resistant cell populations. These programs also emerged in HNSCC patient samples and in relapsed tumors after chemoradiotherapy, linking drug-resistant proteotypes to intra- and inter-tissue spatial heterogeneity. In summary, we describe a robust, versatile and phenotype-resolved approach for uncovering single-cell-derived proteotypes associated with the therapeutic responses of distinct tumor cell clones
Tetrahydrobiopterin enhances regulatory T- and mast cell proliferation and alters cytokines expression in a murine heart transplant model
Get PDFAdministration of tetrahydrobiopterin (BH4) has been shown to attenuate acute allograft rejection in a murine heart transplantation model in a manner similar to that of cyclosporine A. However, its mechanism of action on immune cells remains largely unknown. A fully MHC-mismatched (C3H/He to C57BL/6) mouse heart transplant model was used in this study. The recipients were treated with BH4 or cyclosporine A six days. The degree of acute rejection was assessed by histopathological analysis, splenocytes were analyzed by flow cytometry, and cytokine production was estimated based on the level of protein and RNA in sera and grafts and in vitro in T cell cultures. Proliferation of regulatory T cells and mast cells, suppressor capacity of Tregs, and MLR of T cells were conducted in vitro. Survival curves confirmed the significant improvement observed in the BH4-treated animals. BH4-treatment resulted in a substantial increase in Tregs and mast cells in the secondary lymphoid organs. In vitro assays showed increased proliferation of BH4-treated Tregs and mast cells. Cytokine production in vivo and in vitro in BH4-treated animals revealed an increase in the expression of IL-10, IL-5 and IL-4. BH4-dependent mast cell-derived tryptophan hydroxylase-1 could be excluded as a treatment target in recipient knockout mice. These data suggest that BH4 modulates the innate and adaptive immune systems, resulting in increased proliferation of regulatory T and mast cells accompanied by a modulation of anti-inflammatory cytokines
Interaction of sortilin with apolipoprotein E3 enables neurons to use long-chain fatty acids as alternative metabolic fuel
Get PDFSortilin (SORT1) is a lipoprotein receptor that shows genome-wide association with hypercholesterolaemia, explained by its ability to control hepatic output of lipoproteins. Although SORT1 also shows genome-wide association with Alzheimer disease and frontotemporal lobe dementia, the most prevalent forms of age-related dementias, sortilin’s contribution to human brain lipid metabolism and health remains unclear. Here we show that sortilin mediates neuronal uptake of polyunsaturated fatty acids carried by apolipoprotein E (apoE). Using humanized mouse strains and induced pluripotent stem cell-based cell models of brain lipid homeostasis, we demonstrate that internalized lipids are converted into ligands for peroxisome proliferator-activated receptor alpha inducing transcription profiles that enable neurons to use long-chain fatty acids as metabolic fuel when glucose is limited. This pathway works with apoE3 but cannot operate with the Alzheimer disease risk factor apoE4, which disrupts sortilin’s endocytic activity. Our data indicate a role for the lipoprotein receptor sortilin in metabolic fuel choice in neurons, which may be crucial when glucose supply is limited, such as in the ageing brain