Abstract
During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different ...myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward ‘drop&go’ experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.
Patients with multiple myeloma (MM) routinely receive mRNA-based vaccines to reduce COVID-19-related mortality. However, whether disease- and therapy-related alterations in immune cells and ...cytokine-responsiveness contribute to the observed heterogeneous vaccination responses is unclear. Thus, we analyzed peripheral blood mononuclear cells from patients with MM during and after SARS-CoV-2 vaccination and breakthrough infection (BTI) using combined whole-transcriptome and surface proteome single-cell profiling with functional serological and T-cell validation in 58 MM patients. Our results demonstrate that vaccine-responders showed a significant overrepresentation of cytotoxic CD4
T- and mature CD38
NK-cells expressing FAS
/TIM3
with a robust cytokine-responsiveness, such as type-I-interferon-, IL-12- and TNF-α-mediated signaling. Patients with MM experiencing BTI developed strong serological and cellular responses and exhibited similar cytokine-responsive immune cell patterns as vaccine-responders. This study can expand our understanding of molecular and cellular patterns associated with immunization responses and may benefit the design of improved vaccination strategies in immunocompromised patients.
Background
Mutations in the clonal hematopoiesis of indeterminate potential (CHIP)-driver genes DNMT3A and TET2 have been previously shown to be associated with short-term prognosis in patients ...undergoing TAVR for aortic valve stenosis. We aimed to extend and characterize these findings on long-term outcome in a large cohort.
Methods
A total of 453 consecutive patients undergoing TAVR were included in an up to 4-year follow-up study. Next-generation sequencing was used to identify DNMT3A- and/or TET2-CHIP-driver mutations. Primary endpoint was all-cause mortality. Since CHIP-driver mutations appear to be closely related to DNA methylation, results were also assessed in patients who never smoked, a factor known to interfere with DNA methylation.
Results
DNMT3A-/TET2-CHIP-driver mutations were present in 32.4% of patients (DNMT3A
n
= 92, TET2
n
= 71), and were more frequent in women (52.4% vs. 38.9%,
p
= 0.007) and older participants (83.3 vs. 82.2 years,
p
= 0.011), while clinical characteristics or blood-derived parameters did not differ. CHIP-driver mutations were associated with a significantly higher mortality up to 4 years after TAVR in both univariate (
p
= 0.031) and multivariate analyses (HR 1.429, 95%CI 1.014–2.013,
p
= 0.041). The difference was even more pronounced (
p
= 0.011) in never smokers. Compared to TET2 mutation carriers, patients with DNMT3A mutations had significantly less frequently concomitant coronary and peripheral artery disease.
Conclusion
DNMT3A- and TET2-CHIP-driver mutations are associated with long-term mortality in patients with aortic valve stenosis even after a successful TAVR. The association is also present in never smokers, in whom no biasing effect from smoking on DNA methylation is to be expected.
Graphical Abstract
Multinucleated Reed—Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, ...however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well.
Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC ...self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b(-/-) mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.
In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in ...vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP
mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow-derived cells was sufficient to mimic the increased blood flow recovery of global VASP
mice. In chemotaxis experiments, VASP
neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and β-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP
leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.
Most neurons in the adult mammalian brain survive for the entire life of an individual. However, it is not known which transcriptional pathways regulate this survival in a healthy brain. Here, we ...identify a pathway regulating neuronal survival in a highly subtype-specific manner. We show that the transcription factor Pax6 expressed in dopaminergic neurons of the olfactory bulb regulates the survival of these neurons by directly controlling the expression of crystallin αA (CryαA), which blocks apoptosis by inhibition of procaspase-3 activation. Re-expression of CryαA fully rescues survival of Pax6-deficient dopaminergic interneurons in vivo and knockdown of CryαA by shRNA in wild-type mice reduces the number of dopaminergic OB interneurons. Strikingly, Pax6 utilizes different DNA-binding domains for its well-known role in fate specification and this role of regulating the survival of specific neuronal subtypes in the mature, healthy brain.
► Pax6 is necessary for survival of dopaminergic PGNs ► Loss of CryαA function causes neuronal death upon Pax6 depletion ► Pax6 homeodomain controls expression of CryαA that prevents activation of caspase-3 ► Paired-type homeodomain dysfunction impairs survival of dopaminergic PGNs
CELF6 is a CELF-RNA-binding protein, and thus part of a protein family with roles in human disease; however, its mRNA targets in the brain are largely unknown. Using cross-linking immunoprecipitation ...and sequencing (CLIP-seq), we define its CNS targets, which are enriched for 3′ UTRs in synaptic protein-coding genes. Using a massively parallel reporter assay framework, we test the consequence of CELF6 expression on target sequences, with and without mutating putative binding motifs. Where CELF6 exerts an effect on sequences, it is largely to decrease RNA abundance, which is reversed by mutating UGU-rich motifs. This is also the case for CELF3–5, with a protein-dependent effect on magnitude. Finally, we demonstrate that targets are derepressed in CELF6-mutant mice, and at least two key CNS proteins, FOS and FGF13, show altered protein expression levels and localization. Our works find, in addition to previously identified roles in splicing, that CELF6 is associated with repression of its CNS targets via the 3′ UTR in vivo.
Display omitted
•CELF6 primarily associates with 3′ UTRs of synaptic genes in the mouse brain•CELF6:sequence interaction is assayed using a massively parallel reporter assay•CELF3–6 all result in lower mRNA levels with few changes to translation efficiency•CELF6 targets are derepressed in vivo in Celf6-knockout mice
Rieger et al. assay the function of the RNA-binding protein CELF6 by defining its targets in the brain. They show that CELF6 largely binds 3′ UTRs of synaptic mRNAs. Using a massively parallel reporter assay, they further show that CELF6 and other CELFs are associated with lower mRNA abundance and that targets are derepressed in Celf6-knockout mice in vivo.
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