Type I-E CRISPR-Cas3 represents a genome-editing technology in which large deletions averaging several kilobases are introduced in target regions. However, its genome-editing efficiency varies considerably across targets and cell types, making it difficult to achieve consistent results. Here, we investigated the efficacy and stability of circularized CRISPR RNAs (ccrRNAs) to enhance CRISPR-Cas3-mediated genome editing in human cells. Using in vitro single-strand DNA cleavage assays, we demonstrated that ccrRNA induces Cascade complex formation. Significant genome-editing activity targeting the EMX1 and B2M genes was observed in cellular assays using K562 cells. Long-read sequencing identified large-scale deletion mutations at the target loci and no detectable off-target effects using ccrRNA. Furthermore, ccrRNAs exhibited extended intracellular stability compared with that for linear crRNAs, resulting in an enhanced editing efficiency. These results demonstrate that ccrRNAs enable stable, efficient, and highly specific genome editing and support the broader application of the long-range deletion system.
Anti-TNF therapy is central for treating ulcerative colitis, yet ∼40% are primary non-responders. To find pre-treatment signatures distinguishing anti-TNF non-responders from responders, we did serum cytokine profiling and laser microdissection transcriptomics on paired uninflamed and inflamed colonic epithelium and lamina propria from patients matched at baseline but differing in endoscopic outcome. Although inflammation drove more epithelial changes, the signatures separating non-responders from responders were found in uninflamed lamina propria, with elevated cell-cycle genes and a co-expression network enriched for humoral immune genes consistently higher expressed in non-responders. Cellular deconvolution localized this network to IgA plasma cells, confirmed by tissue staining, and the association was validated by pseudobulk analysis of an independent single-cell atlas. Baseline epithelial signatures or serum cytokines did not differ significantly between groups. These findings point to TNF-independent humoral activity already present in uninflamed mucosa as a candidate contributor to non-response, with implications for prediction and alternative therapies.
Preterm birth is associated with a substantially increased lifetime risk of chronic kidney disease, yet the developmental mechanisms driving this association remain unclear. Using an established mouse model of preterm birth, we demonstrate that premature extrauterine life uncouples nephron progenitor cell (NPC) proliferation from differentiation. Although the nephrogenic window is transiently extended, it fails to rescue nephron endowment. Transcriptomic and biochemical analyses reveal that preterm NPCs mount an immediate stress response, marked by unfolded protein response and endoplasmic reticulum stress signatures. This molecular stress coincides with a disruption of the precise temporal dynamics required for efficient differentiation, predisposing to a permanent nephron deficit. Long-term follow-up confirms that these early defects manifest as adult glomerular hypertrophy, proteinuria, and tubular injury. Our findings identify stress-associated dysregulation of NPC dynamics as a central mechanism linking prematurity to lifelong kidney vulnerability, highlighting the perinatal period as a critical therapeutic window.
Despite the prevalence of the KRAS G12D mutation in approximately 40% of pancreatic ductal adenocarcinoma (PDAC) cases and its association with the poorest prognosis among KRAS variants, the downstream transcriptional effectors driving its aggressive metastatic phenotype remain poorly defined. Through integrated bioinformatics and experimental validation, we identified ARNTL2 as a factor associated with KRAS G12D-mutant PDAC. ARNTL2 directly bound to the SERPINE1 promoter to activate its expression, establishing an ARNTL2-SERPINE1 axis that orchestrated epithelial-mesenchymal transition and drove metastasis in vitro and in vivo. Rescue experiments confirmed SERPINE1 as the indispensable downstream mediator. Clinical validation demonstrated that ARNTL2 and SERPINE1 protein levels correlated positively and stratified patients into distinct prognostic subgroups. Furthermore, this axis altered sensitivity to selective KRAS G12D inhibition in vitro. These findings identify an experimentally supported ARNTL2-SERPINE1 transcriptional axis in KRAS G12D-mutant PDAC that warrants further validation in genotype-faithful and clinically relevant models.
Shape-shifting proteins include intrinsically disordered proteins (IDPs) and fold-switching, or metamorphic proteins. They make up a significant fraction of the “dark proteome” in the protein universe. In this essay, we highlight some often misconceived or underappreciated features of shape-shifting proteins: First, the subtlety of the structure of IDPs; second, the relationship between intrinsic disorder and allostery, third, the possibility of discerning IDP conformational preferences based on energy landscape theory, and fourth, the contributions of disordered regions to fold switching in metamorphic proteins. Lastly, we highlight emerging evidence that fold-switching/metamorphic proteins may contribute to phenotypic plasticity and adaptive evolution by acting as evolutionary capacitors.
Glioblastoma is a devastating brain cancer for which patient survival has remained largely unchanged for decades, underscoring the need for improved disease modeling and analytical tools. Neural stem cells have been identified as cells of origin of glioblastoma, leading to the development of somatic lineage models. Such models have been deeply characterized by sequencing but systematic histological analyses remain limited. Here, we present a multimodal histological characterization of a somatic glioblastoma mouse model. Using 3D light-sheet imaging, we show that the model is highly reproducible and enables quantitative assessment of tumor growth across cohorts. Through multiplex imaging with MACSima™ Imaging Cyclic Staining, we map the cellular landscape and molecular architecture of the tumors and their environments, and provide a curated resource of mouse-compatible antibodies. Finally, we demonstrate that tissue clearing and light-sheet microscopy can be seamlessly combined with multiplex imaging, enabling spatial proteomic characterization of a 3D pre-defined tumor.
Exercise induces adipose tissue activation and physiological cardiac hypertrophy, but the link between adipose tissue activation and cardiac remodeling has not been fully established. Here, we found that three weeks of swim training activated brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) in a PGC1α-dependent manner. Genetic knockout (Adrb2KO) or chemical inhibition of the β2-adrenoceptor (ICI118551) suppressed PGC1α expression and BAT and iWAT activation. To test the role of adipose tissue activation in pressure overload-induced cardiac hypertrophy, abdominal aortic constriction (AAC) surgery was performed in swim-trained Ppargc1afl/fl and adipose-specific knockout of Ppargc1a (Ppargc1aaKO) mice. Compared with Ppargc1afl/fl mice, the protective effect of exercise against pathological remodeling was significantly diminished in Ppargc1aaKO mice, whereas exogenous FGF21 administration recovered it. In summary, this study reveals that swim training activates the ADRB2-PGC1α axis and FGF21 generation in the adipose tissue, which protects against pathological cardiac remodeling.
Local tumor progression (LTP) compromises durable local control after radiofrequency ablation (RFA) for stage I non-small cell lung cancer. We developed a three-dimensional minimum ablation margin (3D MAM) quantification workflow and evaluated its integration with post-ablation CT radiomics for individualized LTP prediction. This dual-center retrospective study included 217 patients divided into training, internal validation, and external validation cohorts. Automated segmentation, deformable image registration, and Visualization Toolkit (VTK)-based analysis enabled quantitative 3D margin assessment. An insufficient MAM (<5 mm) was identified in 147 patients (67.7%). MAM ≥5 mm was independently associated with lower odds of LTP (odds ratio, 0.06; 95% confidence interval, 0.02–0.16). Among nine machine-learning classifiers, the random forest model showed consistent discrimination across cohorts, with areas under the curve of 0.877, 0.851, and 0.889. Combining quantitative margin assessment with CT radiomics may support risk-adapted surveillance after lung RFA.
Heterogeneous nuclear ribonucleoprotein F (hnRNP F), a member of the hnRNP family, is an essential regulator of RNA metabolism, influencing splicing, translation, stability, and telomere maintenance. This review provides a comprehensive overview of the regulation of expression and multifunctional roles of hnRNP F. Owing to the high degree of sequence homology and functional similarity between hnRNP F and hnRNP H/H′, this review also integrates discussions on hnRNP H/H′ to facilitate a comparative and holistic understanding of their biological significance.
An animal’s ability to taste is critical for nutrient selection and rejection of potentially toxic substances. Factors such as age, diet, environment, and internal state can also influence food-related behavioral outcomes. One such factor—exposure to taste cues during early development—remains poorly understood. Here, we evaluate how larval taste input affects adult feeding preference in Drosophila melanogaster. We find that larval dietary exposure to papaverine, a bitter alkaloid, attenuates feeding avoidance of papaverine in adult flies. This behavioral modification appears compound specific, occurring with papaverine but not with other bitter chemicals tested, and selective, with feeding preferences for other bitter tastants unaffected. We identify contributions of larval bitter taste function, taste projection populations, and the cyclic AMP (cAMP)-phosphodiesterase dunce in supporting this behavioral modification. Together, our findings suggest that developmental experiences with bitter compounds can change taste-mediated behaviors through defined sensory and neural circuit components.
Persulfidation is a post-translational modification involving the specific conversion of thiols (-SH) to persulfide groups (-SSH) on cysteine residues in target proteins. This modification plays critical roles in numerous physiological and cellular processes. Previously, we demonstrated that host cell persulfidation levels influence the effectiveness of antifungal immunity against the fungal pathogen Aspergillus fumigatus. In this study, we sought to further elucidate the relationship between persulfidation and the immune response. To this end, we defined the persulfidated proteins (the persulfidome) in alveolar epithelial cells that were challenged with A. fumigatus and/or exposed to a sulfide donor, which increases cellular persulfidation. Our findings suggest that persulfidation indirectly modulates immunity through changes in RNA metabolism and translation. Additionally, calprotectin was persulfidated in response to A. fumigatus exposure, and this modification is essential for maintaining its immune-related function under oxidative stress. Our results suggest that persulfidation positively influences generalized and specific immune responses.
Infection due to the chikungunya virus (CHIKV) can cause chronic disease characterized by persistent arthralgia. While the humoral immune response to CHIKV is well characterized, the contribution of Fc-mediated effector functions in disease pathogenesis remains poorly understood. We assessed the quantitative and qualitative aspects of CHIKV-specific antibodies in patients with self-resolving or chronic chikungunya, including IgG/IgM titers and effector functions such as antibody-dependent cell phagocytosis (ADCP), complement deposition (ADCD), cell cytotoxicity (ADCC), and NK cell activation (ADNKA). Anti-CHIKV IgG and IgM titers declined between 3 and 6 months post-infection, and the lower titers at 6 months were associated with the chronic disease. The ADCP and ADCD capacities were similar between patients with self-resolving or chronic chikungunya. Patients with the chronic disease showed reduced ADCC but no reduced ADNKA. Females exhibited higher anti-CHIKV E2 antibody concentrations and ADCP. This study provides a comprehensive overview of Fc-mediated effector functions in chikungunya patients with differential disease outcomes.
Halide perovskites have demonstrated great potential for next-generation X-ray detectors owing to their strong X-ray absorption, excellent charge-transport properties, tunable band structures, and low-cost processability. These advantages enable high-sensitivity, low-dose, and fast-response X-ray detection for medical imaging, security screening, and industrial inspection. Recently, heterostructure engineering has become an effective strategy for improving detector performance and stability by regulating band alignment, interfacial coupling, and carrier transport. In this review, we highlight recent advances in heterostructure-engineered halide perovskite X-ray detectors, including perovskite/perovskite and perovskite/functional-material heterostructures. Particular emphasis is placed on how heterointerfaces improve charge separation and transport, suppress recombination losses, inhibit ion migration, and enhance device stability and imaging performance. Finally, we discuss the remaining challenges and future opportunities for developing high-performance, stable, and scalable perovskite X-ray detection technologies.
Cancer risk in Latin America is shaped by lifestyle exposures whose regional patterns are usually represented through extrapolated global estimates. We systematically reviewed observational studies on tobacco smoking, alcohol consumption, adiposity, physical inactivity, and dietary patterns and cancer risk in Latin American adults, searching four databases through September 2025 and appraising risk of bias with QUIPS. Forty-seven studies were included, almost all case-control. Pooling was restricted to site-specific sets sharing one exposure contrast, and only two qualified: a higher dietary inflammatory index with prostate cancer (OR 1.38, 95% CI 1.11–1.73) and obesity with endometrial cancer (OR 3.81, 1.08–13.45). In a direction-of-effect synthesis, healthy dietary exposures were protective in 9 of 9 estimates, whereas smoking and unhealthy diets were predominantly associated with increased risk. These associations are directional rather than causal, and they indicate where prospective regional cohorts are most needed.
Approximately 1.5 billion end-of-life tires accumulate globally each year, presenting major environmental and human health challenges. Although recycling pathways such as devulcanization, pyrolysis, and mechanical grinding are promoted to conserve resources, their real-world sustainability is limited by technical, economic, and environmental constraints. These recycling processes can release hazardous pollutants, including volatile organic compounds, polycyclic aromatic hydrocarbons, heavy metals, microplastics, and 6PPD-quinone, posing long-term ecological and toxicological risks. Integrating technical, regulatory, and toxicological perspectives, this review evaluates the risks and benefits of current tire management strategies. It uncovers overlooked downstream impacts and calls for advanced recycling technologies, high-fidelity monitoring systems, and strengthened regulations to enhance the overall sustainability of tire recycling practices.
Carboxypeptidase M (CPM) metabolizes several bioactive peptides by removing their C-terminal arginine residues. It was postulated that the substrates include the anaphylatoxins C3a and C5a generating their less active desArg forms. C3a and C5a are potent mediators of inflammation, shaping both innate and adaptive immune responses. Considering that CPM is a membrane-bound enzyme expressed in multiple organs, we hypothesized that it may protect tissues from anaphylatoxin-driven damage. Using mass spectrometry, we confirmed that CPM efficiently converts C3a and C5a into their desArg forms. We then generated CPM-deficient rats and subjected them to a complement-dependent model of acute lung injury. Compared to controls, CPM-deficient rats exhibited consistently exacerbated lung damage, including higher histological injury scores, increased neutrophil and macrophage infiltration, and elevated expression of inflammatory marker genes. These findings indicate that CPM protects the lung from complement-mediated injury and highlight it as a potential therapeutic target in inflammatory diseases.
Post-stroke neurological decline is associated with persistent vascular and metabolic dysfunction, including long-term alterations in lipid homeostasis. We performed targeted lipidomic analyses in the hippocampus and plasma from rats subjected to transient middle cerebral artery occlusion at 6 h, 24 h, 7 days, 15 days, 1 month, and 4 months after stroke. Lipid profiles were integrated with behavioral, histological, immunofluorescence, western blot, flow cytometric, and bioinformatic analyses. At 4 months, an increased plasma lysophosphatidylcholine/phosphatidylcholine ratio was associated with late neurological deterioration and cognitive impairment. This change coincided with hippocampal lipid remodeling, region-specific astrogliosis, changes in PLA2, LPCAT1, MBOAT1, and SREBF2, and altered circulating BODIPY+ lipophilic particles associated with apolipoproteins. These findings support coordinated central and peripheral lipid remodeling during chronic post-ischemic progression with potential relevance for peripheral biomarker development.
We develop a cluster-level optimization framework to design brownfield transition pathways toward net-zero chemical clusters by 2050, applied to existing ammonia-olefin clusters in the Netherlands. The model represents sequential investment decisions for 2030, 2040, and 2050, includes scope 1–3 emissions, and enables a systematic comparison of greenfield and brownfield configurations. Thirteen olefin and eight ammonia production routes are considered. The results show that, despite reduced flexibility due to legacy assets, net-zero brownfield configurations remain feasible. Plastic gasification combined with methanol-to-olefins emerges as a low-regret option for olefins, while electric steam methane reforming and electrolyzers are robust choices for ammonia. The results support early investment rather than strategic delay. Excluding scope 3 emissions leads to lock-in of fossil-based options that appear cost-effective short-term but raise long-term system costs. Sensitivity analyses highlight uncertainties related to infrastructure and technology availability, bio-feedstock prices, and plastic waste emission accounting.
