Biomolecules

Amyloid fibrils are highly ordered protein aggregates characterized by a conserved cross-{beta}-sheet architecture despite originating from structurally diverse precursor proteins. Growing evidence suggests that interactions between different amyloidogenic proteins can modulate aggregation pathways through heterologous cross-seeding; however, the influence of seed polymorphism on the structure and biological properties of cross-seeded fibrils remains poorly understood. Here, we investigated the cross-seeding of native human insulin by two structurally distinct polymorphs of hen egg-white lysozyme (HEWL): flexible fibrils (FFs) and rigid fibrils (RFs). Native insulin remained stable under physiological conditions and underwent spontaneous fibrillation only under acidic conditions. In contrast, both HEWL polymorphs efficiently induced insulin aggregation at physiological pH, bypassing the nucleation barrier. Thioflavin T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy revealed that lysozyme FFs templated the formation of insulin flexible fibrils (IFFs), whereas lysozyme RFs produced insulin rigid fibrils (IRFs), demonstrating that the structural characteristics of the parental HEWL polymorphs were propagated during heterologous cross-seeding. The toxicity of the resulting insulin fibrils was evaluated in SH-SY5Y neuronal cells and CCF-STTG1 astrocytes. IFFs exhibited minimal cytotoxicity and only subtle morphological alterations, whereas IRFs caused modest reductions in cell viability accompanied by more pronounced cellular damage. These findings demonstrate that the structural polymorphism of HEWL fibrils governs both the architecture and biological activity of cross-seeded insulin fibrils, highlighting amyloid polymorphism as an important determinant of heterologous amyloid propagation and a potential design principle for engineering functional amyloid-based biomaterials and protein delivery platforms.

Background: Serial glial fibrillary acidic protein (GFAP) trajectories have become an important framework for contextualizing evolving secondary-injury pathophysiology after moderate-to-severe traumatic brain injury (msTBI). However, total GFAP pools release and clearance signals that may be less useful for longitudinal bedside decisions than a proteoform-resolved assay. We compared total GFAP with neoGFAP, defined here as calpain-generated GFAP proteoforms intended to index active astroglial proteolysis during the subacute phase. Methods: We analyzed 651 serial serum samples from 95 msTBI patients from a previously described single-site cohort. Total GFAP and neoGFAP were measured on the same MSD platform from 6 to 240 hours after injury. Early (6 to 72 h) and late (96 to 240 h) windows, data-derived tertiles, and serial trajectory summaries were calculated directly from serial samples. Models were benchmarked against age plus admission post-resuscitation Glasgow Coma Scale (GCS) and the admission IMPACT extended risk score using five-fold stratified cross-validation. Outcomes were unfavorable outcome (GOSE 1 to 4), less-than-good recovery (GOSE 1 to 6), Disability Rating Scale (DRS) [≥]15, mortality, and neuroimaging worsening at 6 months. Results: The cohort contributed 95 serial biomarker profiles, with 90 participants evaluable for 6-month GOSE and 89 for DRS. Unfavorable outcome occurred in 57/90 (63.3%), and less-than-good recovery in 79/90 (87.8%). For unfavorable outcome, IMPACT plus early neoGFAP reached AUROC 0.85 versus 0.84 for IMPACT plus early total GFAP and 0.81 for IMPACT alone. For less-than-good recovery, IMPACT plus late neoGFAP achieved AUROC 0.90 versus 0.84 for late total GFAP and 0.82 for IMPACT alone. Secondary analyses for DRS, mortality, and neuroimaging worsening showed smaller differences. Conclusions: In this retrospective analysis, neoGFAP provided clearer incremental value than total GFAP for recovery-oriented monitoring, especially when late-window reassessment of patients who remained at risk for less-than-good recovery was required. Results support prospective testing of neoGFAP as a pathophysiology-informed adjunct to serial bedside decision making, repeat-assessment thresholds, and recovery stratification.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Objectives: Dentists prescribe approximately one in ten antibiotics worldwide, yet antimicrobial stewardship (AMS) remains underemphasized in dental education. Large language models (LLMs) may support AMS training, but their proficiency and clinical reasoning in this context remain unclear. We evaluated GPT-4o's accuracy and clinical reasoning on dental antibiotic prescribing questions, stratified by question difficulty. Methods: We assembled 125 multiple-choice questions on dental antibiotic prescribing from eight peer-reviewed studies (2017-2023). GPT-4o answered each question and generated a clinical justification. Accuracy was assessed against source-study answer keys and examined across difficulty quartiles. Justifications were evaluated using an adapted 12-axis human-evaluation framework assessing scientific consensus, extent and likelihood of harm, inappropriate and missing content, bias, and both correct and incorrect comprehension, retrieval, and reasoning. Prophylaxis-specific questions were analysed separately. Results: GPT-4o correctly answered 72% of questions. Accuracy remained relatively stable across difficulty quartiles (78%, 78%, 65%, 70%). Experts rated 95.4% of justifications positively across the 12 axes. Comprehension, retrieval, and reasoning each exceeded 96.2% positive ratings. Missing content was the main weakness (7.8%), and 7.1% of justifications showed a moderate-to-severe potential for harm. Performance on prophylaxis-specific questions (98.1%) exceeded non-prophylaxis questions (93.0%). Conclusions: GPT-4o demonstrated moderate-to-high proficiency and clinically defensible reasoning in dental antibiotic prescribing questions. However, residual risks indicate that it is not suitable for unsupervised clinical use but shows potential as a supervised AMS educational tool.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

Low-density lipoprotein (LDL) is a major atherogenic lipoprotein, yet its potential to directly modify the fibrin scaffold of blood clots is incompletely understood. Here, we investigated how LDL alters plasma fibrin network architecture and internal fibrinolysis across defined fibrinogen/thrombin conditions. Pooled normal human plasma was supplemented with LDL and clotted with controlled concentrations of fibrinogen and thrombin. Fibrin architecture was visualized by confocal microscopy and quantified by pore-size analysis; clot formation and lysis were monitored turbidimetrically in the presence of tissue plasminogen activator (tPA). Increasing LDL produced a pronounced reduction in fibrin-network pore size across the tested fibrinogen/thrombin conditions. The LDL dependence of pore diameter was well described by a power-law relationship, D_pore=(6.54 +/- 0.11)[LDL]^(-0.12 +/- 0.02) , (R^2 = 0.90), with a significant negative LDL exponent (p = 4 x 10^5). Increasing LDL also prolonged clot lysis time and altered turbidity kinetics. These findings extend epidemiologic and clinical associations between ApoB-containing lipoproteins and hypofibrinolytic clot phenotypes by demonstrating, in a controlled plasma system, that LDL itself can modify fibrin network architecture and fibrinolytic susceptibility. The results support a structure-function role for LDL within the fibrin biomaterial and motivate direct tests of LDL incorporation, protofibril packing, fibrinolytic-protein binding, and single-fiber mechanics.

Carbapenems are the most potent {beta}-lactams, key antibiotics for healthcare-associated infections by Gram-negative bacteria and evade hydrolysis by most {beta}-lactamases, but are increasingly threatened by emergence of enzymes exhibiting hydrolytic activity towards them. Of the four recognised {beta}-lactamase subclasses, class A (active-site serine enzymes that hydrolyse {beta}-lactams via a covalent acylenzyme intermediate) is the most widely disseminated and, while the majority of such enzymes react with carbapenems to form long-lasting acylenzyme complexes, several possess carbapenem-hydrolyzing activity (carbapenemases). Here, we investigate the basis for these differences in a panel of class A {beta}-lactamases using molecular dynamics (MD) simulations of the respective acylenzyme complexes and tetrahedral intermediates (TI). The simulations reveal multiple features associated with catalytic activity across the spectrum of enzymes tested, including more extensive interactions of the carbapenem acylenzyme carbonyl and generally increased lifetimes of active site water molecules positioned for deacylation. Analysis of the dynamic trajectories shows carbapenemases to have reduced root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI, that are not limited to the active site, indicating that the acylenzyme complex is pre-organised for reaction in carbapenemases but not in carbapenem-inhibited enzymes. Similarly, Principal Component Analysis (PCA) of acylenzyme and TI dynamics shows greater overlap between the two states in carbapenemases, providing further evidence for acylenzyme pre-organisation. Such simulations may represent an effective computational assay able to identify enzymes with carbapenemase activity at relatively modest computational cost.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

Metabolic dysfunction-associated steatotic liver disease (MASLD), the leading cause of chronic liver pathologies worldwide, represents a growing clinical burden. Its diagnosis remains reliant on liver biopsy that limits early detection and the ability to capture molecular changes across disease progression. A systematic understanding of stage-dependent gene expression changes is essential to identify biomarkers and effectively characterize disease mechanisms. Therefore recent studies provided databases for searching genes as well as prediction of multi-gene signatures for disease progression. However, there is still a need for interactive and comprehensive meta-analysis of datasets of MASLD patients with available histological metadata. Herein, we performed a meta-analysis of RNA-seq datasets using NAFLD Activity Score (NAS; n = 897) and fibrosis stage (n = 856) upon conducting pairwise comparisons across histological stages and identified differentially expressed genes associated with disease progression. Most importantly, we provide our findings via a dedicated web server, the MASLD-META NETWORK (https://masld.scilicium.com), enabling users to interactively explore meta-analysis results across diverse network modalities. In addition, we characterized gene expression dynamics across increasing disease stages to identify consistent progression-associated pathways using Louvain clustering. Network-based parameters such as centrality in combination with meta-analysis scores further highlighted central genes and pathways implicated in disease mechanisms. Accordingly, MASLD-META NETWORK enabled an integrative reassessment of recently published gene signatures, identifying COL1A1, COL3A1, THBS2, FBLN5, and PDGFA as the most central genes, and SULF2, MMP14, IL32, GPNMB, and COL3A1 as candidate markers of earlier transcriptional alterations. Network analysis of MASLD associated biological modules further identified LAMA2 and LAMA3 as previously unrecognized central candidate targets.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [≥]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

The lack of effective anti-JEV therapy possesses significant challenge to control JEV. {beta}-catenin, a key mediator of Wnt signaling pathway regulates different viral replication and host immune responses. However, its role in JEV infection remains to be elucidated. Thus, the current study focused on evaluating iCRT-14, a specific {beta}-catenin inhibitor, against JEV. Treatment with iCRT-14 following JEV infection resulted efficient reduction in viral progeny release, viral RNA and protein levels in Huh7 and HEK293T cells. Further, active and total {beta}-catenin, Cyclin D-1 and GSK3-{beta}, the other key pathway players were also modulated in infected and inhibitor treated cells. Moreover, iCRT-14 showed an IC of 4.56 in Huh7 cell and maximal inhibition at the early stages of the JEV life cycle. Interestingly, the overexpression of {beta}-catenin in both the cells and siRNA-mediated {beta}-catenin knockdown (in Huh7 cells) significantly abrogated JEV replication, as evidenced by decreased viral titers, viral protein expression, and viral as well as total RNA levels. Moreover, the reduction in extracellular (84%) and intracellular (60%) viral titers following iCRT-14 treatment highlights its role in impairing JEV infection. Further, in silico molecular docking and co-immunoprecipitation studies demonstrated interactions between {beta}-catenin and the JEV NS5 and E proteins. Collectively, these findings suggest that optimum level of {beta}-catenin is required for efficient JEV infection, highlighting its potential as a target for designing host-directed control strategies to regulate viral infection.

Background: Acute phase response is an early immunometabolic response to brain injuries, primarily coordinated by the liver via the activation of acute phase proteins. These immune responses can be both beneficial, promoting tissue repair, and detrimental, exacerbating neurological deficits, if not properly controlled. Despite the central role of the liver in immunometabolism, how hepatic metabolism dynamically adapts to traumatic brain injury remains under explored, primarily due to limited liver-specific modalities that can assess metabolic pathways in vivo. 13C MRI utilizing hyperpolarized 13C-pyruvate can assess key regulatory enzyme activities in hepatic metabolism. Methods: Rats with controlled cortical impact were studied in vivo using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate under fed and fasted conditions 3-4 days after injury. Hyperpolarized 13C products, including [13C]bicarbonate from [1-13C]pyruvate and [5-13C]glutamate, [1-13C]acetyl-L-carnitine, and [2-13C]phosphoenolpyruvate from [2-13C]pyruvate, were evaluated to assess mitochondrial and gluconeogenic metabolism. In parallel, liver tissues were collected following [U-13C3]pyruvate injection for NMR isotopomer analysis of phosphoenolpyruvate, glucose, and glutamate. Results: While no metabolic differences were detected under fed condition, [13C]bicarbonate and [2-13C]phosphoenolpyruvate increased after brain injury under fasted condition, indicating an upregulation of the hepatic gluconeogenic pathway after injury. 13C NMR of liver tissue extracts from injured rats showed an elevated [2,3-13C2]glutamate-to-[4,5-13C2]glutamate ratio and increased 13C-labeling in phosphoenolpyruvate than controls, confirming enhanced hepatic gluconeogenic pathway. Conclusion: This study demonstrates that hepatic acute phase response to brain injuries can be monitored in vivo by hyperpolarized pyruvate, which may be further utilized for longitudinal immunometabolic evaluation of the liver during pathogenesis and therapeutic interventions.

Purpose. Prognostic assessments after acute brain injury are largely narrative, and how prognostic language relates to subsequent care has not been measured at scale. We quantified where it is written and its association with a subsequent code-status limitation. Materials and Methods. Multidatabase observational study of adults with acute brain injury or a related neurologic emergency, using MIMIC-IV (2008-2019; discharge summaries and radiology reports) and a timestamped MIMIC-III cohort (notes and code-status orders). The exposure was documented prognostic language; outcomes were its association with a subsequent full-code-to-limitation transition, note-stream location, and completeness of documented command-following relative to structured Glasgow Coma Scale (GCS) motor scores. Results. Among 31,993 admissions (27,054 patients; median age, 69 years; 54.9% male), prognostic language in the timestamped cohort (MIMIC-III) was associated with a subsequent code-status limitation after multivariable adjustment (adjusted hazard ratio, 4.3; 95% CI, 2.9-6.5; unadjusted 14-day cumulative incidence, 40% vs 8.5%), including the comfort-measures component (3.9), a higher-risk subgroup (4.4), and after acute-physiology adjustment (4.1); the association was concentrated in the first 3 days. Non-prognostic severity language showed no comparable association (hazard ratios, 1.1-1.3). Prognostic language localized almost entirely to the narrative (4.9% of discharge summaries vs 0.015% of radiology reports); command-following was undocumented in 55.7% of summaries, and no final-24-hour GCS motor score was charted in 72.8%. Conclusions. Documented prognostic language after acute brain injury was written in the narrative, not structured fields, and was associated with a subsequent code-status limitation after multivariable adjustment. This observational association cannot establish causation but warrants prospective study.

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.

Background: On-call clinicians frequently report the anecdotal impression of 'theme shifts' during which specific acute neurological diagnoses appear to cluster. Whether such clustering reflects a statistically true and reproducible phenomenon has not been systematically investigated; the present paper examines seasonality and temporal clustering within six different acute neurological conditions. Methods: In this retrospective, single-center cohort study, we identified all patients admitted to a tertiary neurological department between July 2016 and June 2026 with acute unilateral vestibulopathy, cerebral artery dissection, generalized epileptic seizures, primary intracerebral hemorrhage, peripheral facial nerve palsy, or transient global amnesia (TGA) (n = 2,140). Monthly and seasonal distributions were assessed using chi-squared goodness-of-fit and cosinor analysis. Short-term temporal clustering was tested by Monte Carlo permutation across time windows from 24 hours to 90 days, and endogenous cluster dynamics were characterized using Hawkes self-exciting point process modeling. Results: Admissions for generalized epileptic seizures showed a statistically significant deviation from a uniform monthly distribution with a winter distribution (p<0.001 and q = 0.002), and a significant temporal clustering across time windows from 72 hours to 90 days (all q < 0.05). Peripheral facial nerve palsy presented significant clustering at the 90-day window (q = 0.029) and TGA at 60-day time window (q = 0.041) without seasonality; the diagnostic groups of acute unilateral vestibulopathy, cerebral artery dissection and primary intracerebral hemorrhage showed neither seasonality nor clustering after correction for multiple comparison. No diagnostic group showed clustering within a 24-hour window, statistically significant self-excitation in Hawkes process modelling, or a significant linear trend in monthly case counts over the study period. Conclusion: The anecdotal impression of diagnostic 'theme shifts' among on-call neurologists appears to have a measurable basis, although clustering is confined to specific conditions and rather on a time scale of weeks to months. Generalized epileptic seizures were the only diagnostic group that uniquely combined seasonality with temporal clustering, suggesting a shared trigger, while facial palsy and TGA showed episodic, yet non-seasonal clustering.

Initiator-methionine excision is among the earliest protein modifications, yet its relationship to assembly geometry is unknown. Burial of the mature first residue was measured across 7,246 deposited human biological assemblies (22,291 chain-level observations; 1,191 proteins). Among 1,143 analyzable proteins, termini in MetAP-permissive penultimate-residue sequence classes were less often interface-engaged than termini in MetAP-nonpermissive classes (37.4% versus 47.4%; adjusted odds ratio 0.65, p = 7.2e-4). Curated processing annotations did not show a corresponding burial difference, and correlated residue properties preclude attributing the sequence-class association specifically to iMet removal. The analysis identified 264 interface-engaged MetAP-permissive candidates concentrated in cellular machines. In a fully recomputed conformer scan of deeply buried proteasome positions, modeled methionine accommodation was less favorable than at observed-methionine controls (median overlap -0.30 versus -1.12 angstrom, p = 0.0049), although most scoreable sites permitted a nonoverlapping placement. The census therefore reveals a graded structural constraint - not universal steric failure - and prioritizes complexes in which altered packing, assembly kinetics, lipidation or N-terminal methylation can be tested.

Human cytochrome P450 2C9 (CYP2C9) is a hepatic microsomal enzyme involved in the oxidative metabolism of clinically important drugs, but the structural organization of its oligomeric assemblies outside crystallographic packing environments remains poorly understood. Here, we report the cryo-EM structure of human CYP2C9 determined under aqueous, membrane-free conditions at 3.31 Angstrom resolution. The structure reveals a C2-symmetric hexameric assembly organized as a dimer of trimers. Individual protomers retain the conserved P450 fold and heme-binding architecture observed in previously reported crystal structures, indicating that assembly formation does not substantially perturb the catalytic core. The hexamer is stabilized by defined intra-trimer interfaces involving the N-terminal region and residues around Trp212 and Phe482, together with inter-trimer interfaces involving Leu71 and the 220-227 loop. These interfaces are distinct from the crystal packing contacts observed in CYP2C9 crystal structures, demonstrating that the assembly is not a simple recapitulation of crystallographic packing. Notably, the inter-trimer interface is located near the FG-loop-containing surface previously implicated in membrane association. This suggests that the observed hexamer may represent a membrane-free association of two trimers through membrane-related surfaces, whereas the trimeric arrangement itself may be compatible with membrane-associated organization. The structure therefore provides a framework for investigating how trimer formation, membrane interaction and local conformational changes in the FG-loop region may influence CYP2C9 function.

Scientific agents can produce plausible answers while remaining unable to establish whether the computation behind an answer is executable, recoverable, or reproducible. We present BloClaw, an AI4S workstation built around a simple principle: a scientific agent should know what it can do, show how it did it, and state what remains unvalidated. Each capability declares an execution state, input constraints, dependencies, expected outputs, and scientific limitations. Natural-language requests are translated into structured tasks, validated against this registry, executed through scientific tools, and recorded in a provenance-aware Living Lab Notebook. The system is designed to detect invalid inputs, failed tool calls, missing dependencies, and remote timeouts, and to route them to repair, retry, or escalation. The implemented and tested scope comprises RDKit-based molecular property and rule screening, protein structure analysis, docking-pose inspection, 3D visualization, and structured reporting. We demonstrate the workflow on a PubChem-retrieved osimertinib structure and a supplied 6LU7 docking artifact: the former yields deterministic descriptors (molecular weight 499.619 Da, cLogP 4.5098, TPSA 87.55 A^2), while the latter contains 2,387 protein ATOM records, 309 residues, and nine pose records. These examples are workflow demonstrations, not efficacy or affinity studies. Beyond retrospective prediction, the manuscript specifies a prior-minimized constructive mode in which a desired function is compiled into explicit physical, chemical, and systems constraints, candidate mechanisms are simulated, and observations are reintroduced for calibration and falsification; this is a proposed extension rather than a result of the present case studies. We describe an evaluation protocol that compares BloClaw with a standard single-agent workflow and fixed-script execution using task completion, scientific correctness, recovery success, provenance completeness, reproducibility, human review time, latency, and cost. This manuscript reports the system design, verified capability boundary, deterministic software artifacts, and a reproducible evaluation protocol; it does not claim benchmark improvements before those experiments are run. BloClaw is an execution and accountability layer for AI-assisted research, complementing expert review and experimental validation rather than replacing them.