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Penetrant PKCβ mutation in ATLL displays a mixed gain-of-function.
No full textMutations in the T-cell receptor signalling pathway have been identified in patients with adult T-cell leukaemia/lymphoma (ATLL) and one of the most frequently observed targets of these mutations is protein kinase C beta (PKCb). Here we have characterised the most frequent mutation in PKCb (D427N) addressing the issue of gain/loss of function, neomorphic change, assessing the impact of mutation in vivo, in cells, biochemically and structurally. It is concluded that this mutation is a gain-of-function, activating mutation that confers an altered substrate specificity on this protein kinase. In a constitutive knock-in mouse model this activated allele induces splenomegaly associated with extramedullary haematopoiesis. Pharmacologically, the D427N mutant protein displays poor sensitivity to established PKCb inhibitors, necessitating development of bespoke therapeutics for any ATLL intervention through this target. Such efforts could be guided by the availability the D427N mutant-ruboxistaurin structure presented here
Circulating tumor DNA monitoring and blood tumor mutational burden in patients with metastatic solid tumors treated with atezolizumab.
No full textImmune checkpoint inhibitors are important for treatment across tumor types but are not universally effective in controlling disease. Early understanding of tumor response, or lack thereof, can inform treatment decisions. This study evaluates changes in circulating tumor DNA (ctDNA) and blood tumor mutational burden (bTMB) for associations with response to programmed cell death 1 ligand 1 (PD-L1) blockade. We sequenced cell-free DNA collected at the start of therapy, on treatment, and at the end of therapy for 153 patients treated with atezolizumab as part of the pan-tumor MyPathway study (NCT02091141). ctDNA tumor fraction (TF) and bTMB were assessed for correlation with progression-free survival (PFS) and overall survival (OS). We found that molecular response (MR, ≥50% decrease in TF at cycle 3 day 1) was associated with improved PFS (9.7 vs 1.5 months from C3D1; HR = 0.27) and OS (21.1 vs 14.3 months from C3D1; HR = 0.44). These findings were consistent when limited to patients with stable disease (SD; PFS HR = 0.55; OS HR = 0.39). bTMB was correlated with tissue-based TMB (tTMB) when TF was high (≥1%), but not with OS in this cohort. In total, 61% of baseline samples had predicted clonal hematopoiesis (CH) variants. No correlation between maximum variant allele frequency (maxVAF) of predicted CH and TF was seen. In summary, MR is associated with outcomes for patients treated with atezolizumab and could stratify patients with SD. While CH was common, maxVAF for CH variants was not associated with ctDNA TF. Quantification of ctDNA enables therapy response monitoring and is critical for interpretation of bTMB as a proxy for tTMB
Uncertainty of risk estimates from clinical prediction models: rationale, challenges, and approaches
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Sympathetic neurons can modify the intrinsic structural and functional properties of human pluripotent stem cell-derived cardiomyocytes.
No full textThe sympathetic nervous system densely innervates all cardiac chambers and is a key player in cardiac control, yet this relationship has scarcely been investigated using a stem cell-based model. This study investigates the effects that sympathetic neurons (SNs) have on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro, and whether they induce any degree of functional or structural maturity in these conventionally immature cells. SNs were isolated from neonatal rat pups, and cocultured with hPSC-CMs for up to 15 days. Structural changes in hPSC-CMs were analysed by microscopy techniques. Fluorescence resonance energy transfer was used to measure second messenger molecule cAMP production and β-adrenergic receptor (βAR) response. Contractile and Ca2+ transient activity was measured using CytoCypher. These cocultures promoted hPSC-CM structural elongation and increased sarcomere organization. Furthermore, the βAR response of cocultured hiPSC-CMs was larger, indicated by increased cAMP production upon neuronal nicotinic stimulation. Faster contraction and ratiometric Ca2+ transient peak height and kinetic parameters strongly indicate increased chronotropic response in coculture. Coculture with SNs also elicited an increase in action potential amplitude and depolarization velocity, further confirming that SNs contribute to hiPSC-CM functional maturation. Overall, we have found that SNs modulate hPSC-CMs in vitro, inducing a more mature functional response. As an in vitro tool, these cocultures could serve as a model of sympathoadrenergic disease, enabling new discovery avenues. KEY POINTS: The sympathetic nervous system controls the involuntary 'fight-or-flight' response, with the heart being one of key target organs. In certain neuro-cardiac diseases, the input from the sympathetic nervous system is hyperregulated, and can lead to increased speed or force of the heart's contraction. Human induced pluripotent stem cells (hiPSCs) represent a rapidly evolving field which allow us to create a cell of interest and model its structural and functional activity in a dish. Here we have created hiPSC-derived cardiomyocytes (hiPSC-CMs) and cocultured them with sympathetic neurons (SNs). We found that SNs are able to modulate structure of the hiPSC-CMs by reducing their circularity and increasing sarcomeric organization, and can significantly increase the speed of contraction and Ca2+ handling. Together, our data provide a platform to investigate the neuro-cardiac relationship in vitro, which could be used for patient-specific disease modelling in future
ATG9 not just an autophagy related protein.
No full textAutophagy proteins coordinate the biogenesis of a phagophore, the formation and maturation of an autophagosome. Genetic mutations of these proteins can result in dysregulated autophagy, stalled autophagosome biogenesis, and lead to cell death. ATG9, the sole transmembrane ATG (autophagy related) protein governs the nucleation of the phagophore. At a molecular level ATG9 has been shown to be a lipid scramblase capable of redistributing lipids across the lipid bilayer. ATG9-positive vesicles can also deliver lipid-modifying enzymes to alter the lipid composition of membranes. Both functions are required for autophagy. However, ATG proteins, including ATG9, play key molecular roles in pathways unrelated to autophagy. ATG9 has been shown to function in multiple pathways at the Golgi, plasma membrane, and lysosomes. ATG9 can also play an important role in immune signalling. The trafficking of ATG9 in ATG9-positive vesicles is essential to many of these pathways. In this review we highlight the functions of ATG9 in autophagy and autophagy-unrelated pathways, here referred to as "non-canonical functions", and summarise the broader role of ATG9A in cell homeostasis
Impact of statins as immune-modulatory agents on inflammatory markers in adults with chronic diseases: A systematic review and meta-analysis.
No full textWhile numerous studies have extensively documented the pleiotropic effects of statins, including their capacity to reduce inflammation, there is a lack of research estimating the anti-inflammatory effectiveness of statins among individuals with chronic diseases. This meta-analysis evaluates the effect of statin therapy on inflammatory markers and the lipid profile in patients with chronic diseases by analysing evidence from randomized controlled trials (RCTs). We conducted a systematic review and searched articles published between 1st January 1999 and 31st December 2023 in databases including PubMed, Web of Science, Scopus, and Cochrane. The meta-analysis was performed using random effects models and inverse variance. Effect measures were mean differences (MD) and 95% confidence intervals (CI). Collectively, statins significantly reduced IL-6 (MD = -0.24 ng/dL [95% CI, -0.36 to -0.13], I2 = 98.3%, p < 0.001), TNF-α (MD = -0.74 ng/dL [95% CI, -1.08 to -0.40], I2 = 98.8%, p < 0.001); and CRP (MD = -1.58 mg/L [95% CI, -2.22 to -0.94], I2 = 86.5%, p < 0.001). Notably, atorvastatin demonstrated the most significant reduction in IL-6 and TNF-α levels, while fluvastatin and rosuvastatin displayed the greatest impact on decreasing CRP and LDL-C levels, respectively. Stratification by a longer treatment duration of more than four months revealed that atorvastatin achieved the most significant reduction in IL-6 and TNF-α. In conclusion, statin therapy not only regulates the lipid profile but also reduces systemic inflammatory biomarkers. Prolonged administration of statins led to a more substantial reduction in IL-6 and TNF-α, with atorvastatin exhibiting the greatest effect in our analysis
Is the bone marrow microenvironment the hidden catalyst in malignant haematopoiesis?
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Data_EgeaWeiss_TurnerBridger
No full textThe data folder contains all the data needed to generate the figures of the manuscript Egea-Weiss, Turner-Bridger et al. The code to generate the figures is found in https://github.com/Iacaruso-lab/AC-VC-paper/</p
A novel SUN1-ALLAN complex coordinates segregation of the bipartite MTOC across the nuclear envelope during rapid closed mitosis in Plasmodium berghei.
No full textMitosis in eukaryotes involves reorganisation of the nuclear envelope (NE) and microtubule-organising centres (MTOCs). During male gametogenesis in Plasmodium, the causative agent of malaria, mitosis is exceptionally rapid and highly divergent. Within 8 min, the haploid male gametocyte genome undergoes three replication cycles (1N to 8N), while maintaining an intact NE. Axonemes assemble in the cytoplasm and connect to a bipartite MTOC-containing nuclear pole (NP) and cytoplasmic basal body, producing eight flagellated gametes. The mechanisms coordinating NE remodelling, MTOC dynamics, and flagellum assembly remain poorly understood. We identify the SUN1-ALLAN complex as a novel mediator of NE remodelling and bipartite MTOC coordination during Plasmodium berghei male gametogenesis. SUN1, a conserved NE protein, localises to dynamic loops and focal points at the nucleoplasmic face of the spindle poles. ALLAN, a divergent allantoicase, has a location like that of SUN1, and these proteins form a unique complex, detected by live-cell imaging, ultrastructural expansion microscopy, and interactomics. Deletion of either SUN1 or ALLAN genes disrupts nuclear MTOC organisation, leading to basal body mis-segregation, defective spindle assembly, and impaired spindle microtubule-kinetochore attachment, but axoneme formation remains intact. Ultrastructural analysis revealed nuclear and cytoplasmic MTOC miscoordination, producing aberrant flagellated gametes lacking nuclear material. These defects block development in the mosquito and parasite transmission, highlighting the essential functions of this complex
Structural plasticity of Plasmodium falciparum plasmepsin X to accommodate binding of potent macrocyclic hydroxyethylamine inhibitors.
No full textPlasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2'S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S') and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease