Schistosoma mansoni, a causative agent of schistosomiasis, has a complex life cycle that requires extensive molecular adaptation to diverse host environments. Intrinsically disordered proteins (IDPs) and regions (IDRs) are highly flexible protein segments that play key roles in cellular regulation, signaling, and host-pathogen interactions. In this study, we conducted a proteome-wide analysis of IDRs and conditionally folded intrinsically disordered regions (CF-IDRs) in the S. mansoni proteome. 10,749 protein sequences were retrieved from UniProt and reduced to 9638 non-redundant proteins. Intrinsic disorder prediction identified 7662 IDRs, with 36.95% of proteins containing at least one long disordered region. AlphaFold structural confidence scores were integrated with disorder predictions to identify CF-IDRs, defined as disordered regions containing local high-confidence structural segments. Approximately 12.7% of IDR-containing proteins possessed at least one CF-IDR. CF-IDRs were enriched for both disorder-promoting and selected order-promoting residues, suggesting a balance between flexibility and transient structure formation. Prediction of liquid-liquid phase separation (LLPS) propensity revealed that many CF-IDRs may participate in biomolecular condensation. AIUPred-binding prediction indicated that most CF-IDRs contained a strong binding segment, supporting interaction-driven disorder-to-order transitions. Functional enrichment analysis showed that CF-IDR-containing proteins are primarily associated with transcription, RNA processing, chromatin organization, and cytoskeletal regulation. This study is the first to systematically identify and characterize CF-IDRs across the entire proteome of any helminth. Overall, it underscores the importance of structural plasticity in S. mansoni biology and suggests that CF-IDRs may contribute to parasite adaptation, regulation, and host interactions.

Efficient transport of secretory proteins through the endoplasmic reticulum (ER) is essential for their correct localization and cellular function. Misfolded proteins are retained in the ER and eliminated via ER-associated degradation (ERAD), but whether enhanced folding efficiency promotes ER exit and subsequent trafficking remains unclear. To address this question,Arabidopsis carboxypeptidase Y (CPY), a vacuolar protein that passes through the ER, Golgi apparatus, and prevacuolar compartment en route to the vacuole, was used. CPY was fused to three green fluorescent protein (GFP) variants differing in folding properties: standard GFP, superfolder GFP (sfGFP) with enhanced folding capacity, and aggregation-prone GFP[V30A]. As expected, CPY:GFP[V30A] failed to reach the vacuole and accumulated mainly as the precursor form. In contrast, CPY:sfGFP displayed markedly improved vacuolar trafficking compared with CPY:GFP, indicating that enhanced folding efficiency facilitates ER exit and downstream transport. Similar results with CPY95, a truncated CPY variant, further suggest that this effect is independent of protein length. These findings uncover a previously unrecognized role for folding efficiency in regulating secretory protein trafficking, demonstrating that protein folding status can directly influence the efficiency of ER export and vacuolar delivery.

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway and a promising therapeutic target for T-cell-mediated disorders such as leukemia, lymphoma, psoriasis, and rheumatoid arthritis. We used an integrated computational drug-repurposing approach to identify nucleos(t)ide analogs with inhibitory potential against human PNP. A library of approximately 800 nucleos(t)ide analogs underwent machine learning-assisted virtual screening, and the top 20 candidates were further evaluated by molecular docking. The four highest-ranked compounds, based on docking scores (-7.8 to -10.0 kcal/mol), were selected for 500 ns long Gaussian accelerated molecular dynamics (GaMD) simulations. We estimated binding free energies using trajectory-based MM-PBSA calculations and supplemented these with AI-assisted affinity predictions from SG-ML-PLAP and K-DEEP. Among the compounds, 6-hydroxy-flavin adenine dinucleotide (C2) showed the most favorable binding profile, although flavin adenine dinucleotide (C1) had the best docking score (-10.0 kcal/mol). C2 achieved a trajectory-based PBSA binding free energy of -17.23 ± 3.45 kcal/mol, significantly better than the reference inhibitor Immucillin-H (-8.11 ± 2.56 kcal/mol). The other lead compounds, C1, C3, and C4, also demonstrated favorable PBSA binding free energies of -12.90 ± 5.05 kcal/mol, -11.71 ± 3.64 kcal/mol, and -13.19 ± 3.56 kcal/mol, respectively. These results indicate that combining machine learning-assisted screening, molecular docking, enhanced molecular dynamics simulations, MM-PBSA calculations, along with AI-based affinity predictions is an effective strategy for identifying promising nucleos(t)ide analogs for future experimental validation as human PNP inhibitors.

The cyclin-dependent kinase inhibitor p16[Ink4a] is a widely used marker of cellular senescence; however, the contribution of p16[Ink4a]-expressing hepatocytes to hepatic steatosis in metabolic dysfunction-associated steatohepatitis (MASH) remains unclear. Here, we combined p16-CreER-tdTomato lineage tracing, single-cell transcriptomic analysis, and intersectional hepatocyte-specific ablation using a diet-induced MASH model. The NASH2 diet increased the proportion of p16[Ink4a]-lineage-positive [p16-Lin(+)] cells among hepatocytes from approximately 0.4% to 2.0% without significantly altering their proportions among cholangiocytes, endothelial cells, macrophages, or hepatic stellate cells. Single-cell RNA sequencing further showed that hepatocytes constituted 76.5% of the p16-Lin(+) population, compared with 34.4% of the p16-Lin(-) population. Despite their low abundance, selective ablation of p16-Lin(+) hepatocytes reduced hepatic lipid accumulation by approximately 50% (P < 0.0001) without altering the F4/80-positive macrophage area. Hepatocyte re-clustering identified a p16-Lin(+)-enriched hepatocyte subcluster with a transcriptional profile consistent with a zone 2 identity, whereas lipid accumulation occurred predominantly in the pericentral zone 3 region. Within this subcluster, p16-Lin(+) hepatocytes showed increased expression of multiple genes encoding secreted proteins, including complement- and coagulation-associated factors, and the chemokine Cxcl12. Together, these findings demonstrate that a small population of p16-Lin(+) hepatocytes contributes disproportionately to hepatic steatosis, and suggest that secreted factors may mediate their effects on surrounding hepatocytes. Thus, the selective targeting of this population may provide a strategy for limiting hepatic lipid accumulation in MASH.

Bacterial epididymo-orchitis, primarily driven by uropathogenic Escherichia coli (UPEC), is a major cause of inflammatory testicular injury and male infertility. While our previous study indicates that neutrophils are rapidly recruited to the immune-privileged testis to contain the infection, their excessive activation can paradoxically exacerbate tissue destruction. However, the specific cellular states and molecular mediators driving this injurious inflammatory response remain poorly defined. To address this gap, we utilized single-cell transcriptomics in a murine orchitis model to map the dynamic remodeling of the testicular neutrophil compartment. We identified a dominant pathogenic subpopulation (Cluster 0) that rapidly expands during acute infection and is characterized by robust S100a8 expression (accompanied by concomitant induction of its heterodimeric partner, S100a9). Flow cytometric and spatial analyses confirmed the marked accumulation of CD11b[+]Ly6G[+]S100A8[+] neutrophils within damaged reproductive niches. Notably, pharmacological inhibition of the S100A8/S100A9 complex with paquinimod reduced this pathogenic neutrophil infiltration and preserved testicular architecture. Together, these findings identify S100A8 as a key marker and candidate contributor to neutrophil-mediated tissue injury, and suggest that this axis warrants further investigation as a targeted immunomodulatory strategy that may help preserve fertility during bacterial infections.

Diabetic peripheral neuropathy (DPN) is associated with oxidative stress induced by hyperglycemia. Ferritinophagy is critical for maintaining intracellular iron homeostasis and oxidative injury induced by iron overload. However, the relationship between ferritinophagy and high glucose-induced neuronal oxidative damage has not yet been explored. In this study, we established a model of high glucose-induced oxidative damage in ND7/23 cells. ND7/23 cells treated with rapamycin or bafilomycin A1 were used to investigate changes in ferritinophagy, while cells transfected with NCOA4-siRNA were employed to explore the role of ferritinophagy in this process. The results revealed that hyperglycemia inhibited the viability of ND7/23 cells, increased intracellular iron overload, ROS accumulation, and triggered abnormal ferritinophagy characterized by elevated ferritin, NCOA4, and p62 protein levels, together with a reduced LC3II/I ratio. Activation of ferritinophagy with rapamycin alleviated high glucose-induced iron overload and oxidative injury in ND7/23 cells. In contrast, inhibition of autophagy by bafilomycin A1 exacerbated these high glucose-mediated damages. Intriguingly, we found that NCOA4 knockdown improved high glucose-impaired ferritinophagy, reduced intracellular iron concentration, and mitigated oxidative damage in ND7/23 cells treated with high glucose. In conclusion, we conclude that high glucose inhibits ferritinophagy, disrupts iron homeostasis, thereby leading to intracellular iron accumulation and neuron oxidative stress injury in ND7/23 cells. Our results may provide novel perspectives and potential strategies for the prevention and treatment of high glucose-induced oxidative damage.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall and remains the leading pathological basis for myocardial infarction and ischemic stroke worldwide. Despite advances in imaging and risk-prediction models, early detection of subclinical atherosclerosis and identification of plaque vulnerability remain challenging. Conventional biomarkers such as low-density lipoprotein cholesterol, triglycerides, and high-density lipoprotein cholesterol provide valuable risk estimates but lack sufficient sensitivity and specificity for early disease detection and plaque vulnerability assessment, prompting growing interest in non-traditional and emerging biomarkers, as well as instrumental surrogate markers such as pulse wave velocity, which better reflect the complex molecular and mechanical processes underlying atherogenesis. This review provides a comprehensive, mechanism-based overview of traditional and emerging biomarkers associated with atherosclerosis, including inflammatory mediators, coagulation factors, cardiac-specific proteins, adhesion molecules, microRNAs, extracellular vesicles, and novel cytokines, alongside a comparative appraisal of the major non-invasive, functional, invasive, and nuclear imaging modalities used for plaque detection and characterization. Particular emphasis is placed on recently identified biomarkers such as midkine, pentraxin-3, soluble ST2, growth differentiation factor-15, and myeloperoxidase, including their tissue-specific origins and interpretive limitations, which show promise in improving early diagnosis, risk stratification, and prognostic evaluation. Biomarkers are further mapped to the key pathological stages of atherogenesis, endothelial dysfunction, lipid retention and oxidation, vascular inflammation, immune cell activation, plaque destabilization, and thrombotic activation, offering a physiologically intuitive framework for interpreting multi-marker panels. Understanding the biological relevance and clinical utility of these biomarkers may facilitate more precise cardiovascular risk assessment and support the development of targeted preventive and therapeutic strategies.

The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the master circadian pacemaker that synchronizes internal rhythms with the environmental light-dark (LD) cycle. Although SCN spike activity is crucial for maintaining circadian rhythms, there is a scarcity of long-term in vivo electrophysiological recording data, especially for ultradian cycles. Using long-term extracellular single-unit recordings in anesthetized rats, we discovered that multiple types of ultradian cycles, which beyond the three previously reported types, can coexist within a single SCN neuron. The period of these ultradian cycles in one single neuron may shift from nearly 2 min to around 10, 30 or around 80 min, or vice versa. These dynamic changes suggest that periodic stimuli may be particularly suitable for probing the electrical characteristics of SCN neurons. We therefore applied periodic light stimulation in anesthetized rats and performed spectral analysis for neuronal activity. We found that SCN neurons can be entrained by short-period LD cycles ranging from seconds to minutes. Notably, the induced oscillatory firing patterns persisted for more than 1.5 h after stimulus cessation, suggesting persistent reverberatory activity within SCN circuitry. The correlation between firing during and after stimulation increased with stimulus duration and cycle number, suggesting that stronger or longer LD exposure may reinforce post-stimulus oscillatory activity. Moreover, light-on cells exhibited more robust post-stimulus oscillations than light-off cells, implying potentially differential roles in photic encoding. These results suggest periodic light changes can induce persistent rhythmic electrical activity within SCN neurons, potentially reflecting a previously unrecognized form of sub-circadian plasticity within the SCN. We propose an "incomplete gear" model as a hypothetical framework for interpreting temporal dynamics in the SCN. In this framework, SCN neurons exhibit variable ultradian cycle periods, which can be conceptualized as a "incomplete gear" with heterogeneous tooth pitches. Periodic light exposure may bias SCN neuronal activity toward shorter or longer ultradian cycles, and the accumulation of these shifts could contribute to changes in the overall periodicity of neuronal activity, potentially contributing to broader temporal reorganization within SCN networks.

Persistent inflammation, polymicrobial biofilm colonization, and impaired re-epithelialization characterize the chronic and treatment-resistant nature of diabetic wounds. Conventional treatment strategies often fail to adequately address the multifactorial pathophysiological mechanisms underlying these lesions. This review examines the biological functions, key components, and design principles of multifunctional hydrogel nanobiocomposites for diabetic wound healing. These hybrid systems combine natural biopolymers with functional nanoparticles specifically engineered for diabetic wound management. Such nanobiocomposite hydrogels provide an integrated platform capable of simultaneously controlling infection, modulating inflammation, and promoting tissue regeneration. Furthermore, the incorporation of bioactive agents, such as peptides and polyphenols, together with metallic nanoparticles, plays a critical role in enhancing therapeutic efficacy. Finally, this review discusses the major challenges associated with the clinical translation of these systems, including regulatory considerations, standardization, and large-scale manufacturing requirements, while delineating an actionable roadmap for their clinical integration.

Extracellular matrix-derived mechanical signals play an important role in maintaining chondrocyte homeostasis, and their disruption has been implicated in osteoarthritis progression. Here, we investigated whether the long non-coding RNA NORAD reflects substrate stiffness-associated phenotypic alterations in human chondrocytes. Immortalized and primary human chondrocytes were cultured on substrates with defined stiffnesses ranging from 0.2 to 64 kPa or on tissue culture plastic as a rigid control. NORAD expression was differentially regulated according to substrate stiffness and chondrocyte phenotypic conditions. Primary human chondrocytes cultured on intermediate-stiffness substrates at 32 and 64 kPa showed better preservation of the chondrogenic phenotype, reduced phenotypic instability, and attenuated TNF-α-induced inflammatory and hypertrophic responses at the mRNA level. In late-passage dedifferentiated chondrocytes, stiffness modulation partially rescued the loss of chondrogenic marker expression and was accompanied by altered NORAD expression. These findings suggest that NORAD expression reflects stiffness-associated changes in chondrocyte phenotype and may help evaluate mechanically defined culture conditions for chondrocyte maintenance.