Interindividual variability in response to aspirin remains a major challenge in antiplatelet therapy. Identification of patients with high platelet reactivity (HPR) and optimal or low platelet reactivity remains challenging and may be influenced by both biological and methodological factors, including interlaboratory variability. We report results of a large cohort of aspirin-treated patients to assess the gold standard light transmission aggregometry (LTA). We retrospectively analyzed 3065 patients treated with aspirin (ASA; 75-150 mg/d) who sequentially underwent LTA assessment using multiple agonists (adenosine diphosphate [ADP] 2μM, collagen 2 μg/mL, arachidonic acid [AA] 0.75mM, and epinephrine 10μM). Patients were stratified according to AA-induced platelet aggregation using a 16% cutoff (third quartile). Patients with AA >16% showed significantly higher platelet aggregation across all agonists (P < .0001). However, a substantial proportion of patients with AA ≤16%, consistent with effective COX-1 inhibition, still exhibited HPR when stimulated with ADP, epinephrine, or collagen. Collagen-induced aggregation was particularly informative in identifying residual platelet reactivity. Sex-related differences were observed, with female patients showing HPR, especially to ADP and collagen. Platelet reactivity in aspirin-treated patients is highly heterogeneous, with hyperreactive or hyporeactive phenotype potentially affecting the safety and efficacy of antiplatelet therapy. AA-induced aggregation alone is insufficient to fully characterize platelet responsiveness. A multiagonist approach, particularly including collagen, may improve identification of patients with residual platelet reactivity and support personalized antiplatelet strategies. These data support ongoing strategies of guided selection of antiplatelet therapy in patients with cardiovascular disease.

Accumulation of pre-mutagenic 8-oxoguanine and impaired DNA repair can lead to poly(ADP-ribose) (PAR) accumulation and parthanatos in Alzheimer's disease, the leading cause of age-dependent dementia. However, the occurrence of parthanatos has not been well established in other types of dementia, including juvenile neuronal ceroid lipofuscinosis (JNCL), a major form of juvenile dementia. In this study, we investigated parthanatos-associated pathology in JNCL using patient-derived lymphoblasts (PDLs). Quantitative immunofluorescence microscopy revealed increased accumulation of 8-oxoguanine and PAR in JNCL-PDLs. JNCL is known to exhibit elevated intracellular calcium, which activates PAR polymerase - 1 (PARP1). The L-type calcium channel blocker diltiazem effectively suppressed PAR accumulation in JNCL-PDLs, even under co-treatment with a calcium ionophore. Diltiazem also reduced 8-oxoG accumulation in JNCL-PDLs, although this effect was largely calcium independent. Subcellular fractionation followed by immunoblotting showed that diltiazem enhanced nuclear recruitment of OGG1 and its partner proteins. These protective effects were abolished by OGG1 knockdown, suggesting a dual role of diltiazem in regulating both calcium signaling and OGG1 activity. Collectively, our data identify that parthanatos as novel therapeutic target for JNCL and propose diltiazem as a potential therapeutic agent. KEY MESSAGES: Increased poly(ADP-ribose) accumulation represents a novel therapeutic target in juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations. Increased PARP1 expression and decreased PARP1 cleavage lead to elevated poly(ADP-ribose) accumulation in juvenile neuronal ceroid lipofuscinosis. Diltiazem ameliorates poly(ADP-ribose) accumulation in patient-derived cell models of juvenile neuronal ceroid lipofuscinosis by suppressing PARP1 expression and 8-oxoguanine accumulation.

Summary: Background: Cardiovascular physiology exhibits circadian rhythms, including a morning surge in platelet reactivity and cardiovascular events. Aligning the timing of antiplatelet therapy with these rhythms may be a simple, cost-effective approach to improve clinical efficacy. This systematic review evaluates the diurnal variability in platelet reactivity among individuals receiving aspirin, clopidogrel, ticagrelor, and prasugrel and explores the potential for timed administration to optimise platelet inhibition and other surrogate cardiovascular markers. Patients and methods: PubMed, Embase, Cochrane Central Register of Controlled Trials, and trial registries (e.g., ClinicalTrials.gov) were searched from 1980 to August 2025 for randomised controlled trials and observational studies in adults investigating morning versus evening dosing or diurnal efficacy. Results: Of 2,340 records, 25 studies were included (17 on aspirin, seven on clopidogrel, one on ticagrelor, and three overlapping with prasugrel). For aspirin, the data showed a tendency that evening administration achieved greater reductions in morning Cyclooxygenase-1 (COX-1)-dependent platelet reactivity in healthy volunteers and cardiovascular disease patients, although there were mixed effects on ambulatory blood pressure. Clopidogrel exhibited diurnal variability, with peaks in Adenosine Diphosphate (ADP)-induced aggregation occurring in the morning, while prasugrel provided more consistent inhibition than clopidogrel or ticagrelor. No studies directly compared evening versus morning dosing for P2Y12-Inhibitors, and clinical outcomes such as major adverse cardiac events were only assessed in one included study. Conclusions: While evening aspirin may enhance chronopharmacological efficacy, the available evidence is limited due to heterogeneity and variable quality. The limited number of studies on agents beyond aspirin highlights a critical research gap, with robust Randomised Controlled Trials needed to evaluate clinical benefits and inform chronotherapeutic strategies.

Coagulopathy after traumatic brain injury (TBI) is multifactorial and dynamic, with major implications for hemorrhagic progression and outcome. Platelet dysfunction is an underrecognized component that may occur despite preserved platelet count. For the purpose of this point of view, platelet dysfunction refers to any measurable impairment in agonist-induced aggregation, receptor-pathway responsiveness [adenosine diphosphate (ADP)/P2Y12 and arachidonic acid (AA)/ cyclooxygenase-1 (COX-1) axes], or platelet contribution to whole-blood-clot strength, occurring disproportionate to or in the absence of thrombocytopenia. Functional impairment can appear early and has been associated with TBI severity, intracranial hemorrhage progression, transfusion requirements, and mortality. Conventional coagulation tests and platelet count are insufficient to identify this phenotype. Platelet function assays, including thromboelastography (TEG) with platelet mapping, may help characterize selected high-risk patients. However, the evidence remains largely observational, thresholds are heterogeneous, interassay concordance is poor, and platelet transfusion or desmopressin should not be regarded as routine interventions. This point of view reframes platelet dysfunction after TBI as a dynamic hemostatic phenotype and argues for prospective phenotype-guided trials.

Malignant hyperthermia (MH) is a pharmacogenetic condition triggered by volatile anesthetics, which activate MH susceptible mutants of ryanodine receptor 1 (RyR1). The precise binding sites and mechanisms of the therapeutic dantrolene and MH triggers have remained unclear. Here, we present structures of RyR1 bound to dantrolene and the MH trigger 4-chloro-m-cresol (4CmC), revealing the dantrolene and 4CmC binding sites in atomic detail. Dantrolene binds stacked with ATP or ADP in the RY12 domain at the corner of the receptor, inducing a conformational change. Functional analyses confirm ATP or ADP is required for dantrolene inhibition, and a single point mutation disrupting this peripheral ATP site abolishes this ATP/ADP-dependent inhibition. Strikingly, in the absence of dantrolene, this site selectively binds two ADP molecules even in the presence of excess ATP, indicating a role in ATP/ADP ratio sensing. Additionally, we identified four 4CmC binding sites, revealing one primary binding site in an amphipathic cavity in the core solenoid, and three auxiliary binding sites in the EF-hand, transmembrane, and RY12 domains. These findings offer insights into the binding modes and mechanisms of MH-linked molecules, and reveal the RY12 domain's regulatory role as a promiscuous sensor for adenine nucleotides and small ligands.

TheThe colonization of the gastric epithelium with Helicobacter pylori elicits a robust nuclear factor kappa-light chain enhancer of activated B cells (NF-κB)-dependent inflammatory response. The bacterial effector molecule adenosine diphosphate-glycero-β-D-manno-heptose (ADP-heptose) triggers both the classical and alternative NF-κB pathways almost simultaneously leading to activation of the transcription factor heterodimers p50/RelA and p52/RelB. Previous studies have demonstrated that the COP9 signalosome complex (CSN) plays an important role in regulating the classical NF-κB pathway through deneddylation of the E3 ligase Skp1-Cullin1-F-Box-β-TRCP (SCFβ-TRCP), while the influence of the CSN on the alternative NF-κB pathway remains unclear. Here, we show that CSN-dependent deneddylation of Cullin-1 controls via the classical NF-κB signaling pathway the upregulation of RelB mRNA and protein expression, enhancing the nuclear translocation of RelB. These findings are supported by the fact that ADP-heptose induces RelB expression in 2D mucosoids derived from 3D organoids from gastric antral glands of the stomach. Blocking neddylation and the activity of SCFβ-TRCP by treatment with the NEDD8-activating enzyme inhibitor MLN4924 prior to H. pylori infection, stabilized IκBα as well as p100 and suppressed RelA and RelB nuclear translocation. Therefore, our data show that CSN-dependent regulation of SCFβ-TRCP controls classical NF-κB signaling and consequently contributes to RelB nuclear translocation and alternative NF-κB activity. For the termination of alternative NF-κB activity, the CSN serves as a hub between constitutively associated c-Cbl and nuclear RelB, allowing c-Cbl-mediated ubiquitinylation of nuclear RelB following H. pylori infection.

Genome instability (GI) is a hallmark of cancer. GI is associated with accumulative DNA damage and tumor-specific defects in DNA repair. Multiple antitumor drugs have been developed to promote persistent GI and to drive DNA damage beyond a threshold that tumor cells can survive. However, regulatory roles of circular RNAs (circRNAs) in GI and DNA damage remain elusive. Through circRNA profiling and a small interfering RNA (siRNA)-mediated screen, we have identified a circRNA termed circRBM39(4,5,6), which is significantly decreased in breast cancer (BC) and disrupts DNA damage response. CircRBM39(4,5,6) inhibits breast tumorigenesis through increasing DNA damage and promoting sensitivity to the antitumor drug poly Adenosine Diphosphate (ADP-ribose) polymerase inhibitor (PARPi). Mechanistically, cytoplasmic circRBM39(4,5,6) interacts with replication protein A2 (RPA2), an essential DNA repair factor, through a 7-nt AG-rich motif and destabilizes RPA2 protein by facilitating HECT and RCC1-like domain 2 (HERC2)-mediated ubiquitination, thus repressing homologous recombination repair. Importantly, administration of in vitro synthesized circRBM39(4,5,6) substantially inhibits breast tumorigenesis, sensitizes BC cells to PARPi, and renders them susceptible to synthetic lethality (SL) in distinct breast tumorigenesis mouse models. Our findings highlight the interplay between circRNA and GI in cancers, and the SL of circRBM39(4,5,6) to PARPi provides strategies for RNA-based BC therapeutics.

Phosphoketolases (Pkts) naturally catalyze thiamine diphosphate (TPP)-dependent C-C bond cleavage in phosphorylated ketoses through formation of a covalent TPP-bound intermediate, yielding an aldose phosphate and acetyl phosphate (AcP). Since the reaction mechanism does not directly involve the substrate-bound phosphate group, Pkts can be engineered towards efficient synthesis of the high-energy phosphoryl donor AcP from non-phosphorylated substrates in presence of inorganic phosphate. Industrial interest arises from the applicability of engineered Pkt variants in cell-free reaction systems for ATP regeneration from inexpensive substrates such as d-fructose. This chapter describes engineering of the fructose-6-phosphate phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) towards improved activity on d-fructose, with a focus on the establishment of a high-throughput screening (HTS) system for accelerated identification of variants with optimized catalytic performance. Detailed protocols are provided for the expression of pkt libraries in 96-well microtiter plate format and screening for enhanced activity on d-fructose. Furthermore, production of selected variants in shake-flask cultures, enzyme purification and activity assays for validation of hits are covered. The chapter additionally outlines the demonstration of cell-free ATP-regenerating enzyme cascades using engineered Bad.F6Pkt variants. In previous work, application of this workflow enabled identification of the Bad.F6Pkt H548N variant, exhibiting a 5.6-fold increased activity on d-fructose compared to the wild-type. When applied in an ATP-regenerating cascade combined with glycerol kinase, acetate kinase and erythrulose-isomerase activities, this variant enabled ATP yields of up to 2.53 molATP molFructose[-1]. The strategy is broadly applicable and can be adapted for screening Pkt libraries towards increased activity on other non-phosphorylated substrates.

Energy metabolism matches adenosine triphosphate (ATP) resynthesis to hydrolysis even during abrupt transitions such as exercise onset. In 1984, Alois Mader proposed a mechanistic model that captures this regulation through 33 coupled differential equations linking oxidative phosphorylation, glycolysis, and the phosphocreatine shuttle via adenosine diphosphate (ADP), phosphate, and pH feedback. Despite its potential, broader adoption has been limited by closed-source implementations and key publications available only in German. Here, we present MetaboliSim, an open-source software tool that simulates muscle energy metabolism in a virtual human parameterized by four physiological inputs: V̇O2max (oxidative capacity), vLamax (glycolytic capacity), body mass, and active muscle mass. Using MetaboliSim, we test Mader's equations against 10 experimentally established phenomena of exercise metabolism, including ATP homeostasis under fatigue, transient pH alkalinization, glycogen-dependent lactate thresholds, intensity-dependent fat oxidation, the V̇O2 slow component, and work-rate-dependent exhaustion, without altering any equation or constant within the model. Mader's model qualitatively reproduces all 10 phenomena, generating similarly shaped response curves; absolute values depend on individual parameterization rather than being universally matched. The characteristic inverted-U of fat oxidation and the exercise intensity-dependent V̇O2 kinetics, for example, both emerge from the same equation system. A Sobol sensitivity analysis (S01) reveals a calibration hierarchy: V̇O2max and body mass drive power predictions; vLamax and buffer capacity drive metabolite predictions. That a compact, 40-year-old equation system accounts for this breadth of metabolic phenomena suggests that a small number of regulatory feedback loops may suffice to explain a wide range of metabolic responses to exercise. MetaboliSim is freely available to enable independent testing, empirical calibration, and refinement.

The high value of surgery aimed at macroscopic complete resection is undisputed. The TRUST trial (Trial of Radical Upfront Surgical Therapy) aimed to determine the ideal timing of surgery (primary vs. interval debulking). While a significant progression-free survival benefit for primary surgery was demonstrated, the overall survival benefit was only numerical and not statistically significant. Based on the TRUST trial, primary surgery should still be recommended if macroscopic complete resection can be achieved with acceptable morbidity. Careful patient selection at specialized centers is crucial. A milestone in the systemic treatment of advanced ovarian cancer is the option of maintenance therapy with bevacizumab, poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors, or the combination of olaparib plus bevacizumab. Biomarker testing, specifically BRCA and homologous recombination deficiency (HRD) testing, is essential for treatment selection. In case of recurrence, where platinum-based chemotherapy is an option, platinum-containing combination chemotherapy should be administered, and the use of maintenance therapy should be considered. If platinum is not an option, three phase III trials have fortunately now demonstrated a significant overall survival benefit with the antibody-drug conjugate (ADC) mirvetuximab soravtansine (MIRV) in patients with high α‑folate receptor expression (MIRASOL trial), or with pembrolizumab plus paclitaxel ± bevacizumab in patients with CPS ≥ 1 (KEYNOTE-B96 trial), as well as with relacorilant plus nab-paclitaxel (ROSELLA trial), each compared to standard chemotherapy. MIRV and Pembrolizumab are already approved. The increasingly complex treatment of ovarian cancer should therefore be carried out in specialized German Cancer Society (DKG)-certified gynecological cancer centers as an effective tool for quality improvement.