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Background/Objectives: Previous studies have demonstrated the safety of pre-seasonal treatment with the mannan-conjugated birch pollen allergoid EP-088-T502. However, the safety of a combined pre- and co-seasonal treatment regimen has not yet been investigated. As climate change is associated with earlier and less predictable onset of birch pollen seasons, planned pre-seasonal allergen immunotherapy may unintentionally overlap with natural pollen exposure. Therefore, evaluation of the safety of treatment administered during the pollen season is of increasing clinical relevance. This study aimed to compare, in a purely descriptive manner, the safety and tolerability of pre-seasonal versus pre- and co-seasonal treatment with EP-088-T502. Methods: In this prospective, open-label, phase III trial (T502-SIT-059) (EudraCT No.: 2022-004082-20), patients (N = 109) who had participated in a preceding pivotal phase III study were offered continuation treatment with active EP-088-T502 (10,000 mTU/mL) across five treatment visits. For the subgroup analysis, all patients who completed their last treatment visit before 9 April 2023 (and, thus, before the start of the birch pollen season in Germany) were assigned to the pre-seasonal group (N = 20). Those who performed the last treatment visit thereafter were assigned to the pre-/co-seasonal group (N = 83). Due to post hoc subgroup allocation and unequal subgroup sizes, all subgroup analyses were purely descriptive. Results: No deaths nor serious adverse events (SAEs) were reported during the study. No epinephrine administration was required. Systemic adverse drug reactions (SADRs, N = 3) occurred in two patients who had previously received placebo. No grade III or IV systemic reactions, according to the German AWMF classification, were observed. Patients receiving pre- and co-seasonal treatment developed smaller wheals (mean diameter) compared with the pre-seasonal group (immediate reactions: 0.6 vs. 0.7 cm; late-phase reactions: 0.3 vs. 0.4 cm at the last treatment visit). This was also reflected in the medians (immediate reactions: 0.2 cm vs. 0.4 cm; late-phase reactions: 0.2 vs. 0 cm at the last treatment visit). Of all AEs that were (possibly) related to EP-088-T502 (N = 89), 74 (83%) occurred at the first three treatment visits (before the birch pollen season). The frequency of AEs appeared descriptively similar between groups for the last two treatment visits. Patients who had received placebo in the previous trial experienced more treatment-related side effects compared to patients who had already received EP-088-T502 in the previous year. Conclusions: These data suggest that EP-088-T502 is safe and well-tolerated, even when administered during the birch pollen season, regardless of prior exposure to EP-088-T502.

African swine fever virus (ASFV) and porcine epidemic diarrhea virus (PEDV) differ in viral biology and cellular tropism, yet both pathogens suppress macrophage-mediated immune responses in pigs. To identify a conserved macrophage suppression module shared by ASFV and PEDV and evaluate quantum computing as an independent framework for biological network validation. Integrated analysis of publicly available GEO datasets (GSE231435 for ASFV and GSE306895) identified 471 shared downregulated genes. A network- and multi-omics-informed 20-gene core was selected and encoded as a 20-qubit modularity-based Quadratic Unconstrained Binary Optimization (QUBO) problem. Community detection was benchmarked using the Quantum Approximate Optimization Algorithm (QAOA) on both the IBM Quantum Aer simulator and the 156-qubit IBM Fez (Heron r2) quantum processor and compared with brute-force enumeration and simulated annealing. A conserved macrophage suppression module shared by ASFV and PEDV was identified. For the STRING protein-protein interaction network, QAOA at circuit depth p = 3 reproduced the brute-force optimum with an approximation ratio of 1.000. In contrast, performance progressively declined in the denser co-expression network with increasing circuit depth, consistent with noise accumulation under current Noisy Intermediate-Scale Quantum (NISQ) conditions. Multi-run consensus analysis identified stable hub genes, including MMP9 and SLA-DOA, as well as genes exhibiting variable community assignments. These findings reveal a conserved macrophage suppression module shared between ASFV and PEDV and demonstrate that quantum computing can serve as an independent validation framework for biologically meaningful host-response networks. Network topology emerged as a key determinant of QAOA performance on real NISQ hardware.

Immune checkpoint signaling and regulatory T cells (Tregs) play essential roles in immune homeostasis and immune dysregulation in autoimmune diseases. Programmed cell death-1 (PD-1) signaling and Treg dysfunction have been implicated in systemic lupus erythematosus (SLE); however, temporal variation in immune regulatory profiles across disease states remains incompletely characterized. This study aimed to characterize PD-1 expression and Treg-associated immune profiles in patients with SLE compared with healthy controls while descriptively exploring selected illustrative clinical immune contexts. An exploratory longitudinal immunophenotyping study was conducted using multiparameter flow cytometry. The primary exploratory cohort included healthy Taiwanese controls (n = 20) and patients with SLE (n = 10). PD-1 expression across representative T-cell subsets and distributions of Treg-associated immune populations were assessed. Where available, paired longitudinal samples obtained during clinically active and stable disease states were evaluated descriptively to characterize temporal immune variation. Additional illustrative clinical cases representing infection-related and postoperative inflammatory conditions were retained separately for contextual interpretation. Because of the exploratory design and modest sample size, all statistical analyses were considered exploratory and hypothesis-generating. Compared with healthy controls, patients with SLE demonstrated broader inter-individual variation in PD-1 expression and Treg-associated immune profiles, including differences in activated and memory-associated Treg phenotypes. Paired longitudinal evaluation suggested temporal immune variation between clinically active and stable disease states in selected individuals, although substantial heterogeneity remained evident across patients. Healthy controls also demonstrated measurable baseline variability in immune marker distributions. Selected illustrative clinical cases demonstrated heterogeneous checkpoint-associated and regulatory immune profiles under infection-related and postoperative inflammatory conditions. This exploratory longitudinal immunophenotyping study describes variation in PD-1 expression and Treg-associated immune profiles across healthy individuals and patients with SLE while providing descriptive contextual observations under selected inflammatory and postoperative clinical conditions. Because of the exploratory design, modest cohort size, clinical heterogeneity, and absence of functional immune validation, all findings should be interpreted cautiously and considered exploratory and hypothesis-generating rather than confirmatory. Larger longitudinal studies incorporating standardized immune monitoring and functional immune assessment are warranted to clarify the biological and clinical relevance of these preliminary observations.

Materials enabling the cell-responsive delivery of endogenous biologics, such as growth factors, have the potential to modulate wound repair cost-effectively and safely. Unlike passive drug delivery strategies that require supraphysiological doses of recombinant protein or stimuli-responsive systems that rely on external triggers, we demonstrate a strategy that harnesses cellular traction forces as an intrinsic delivery trigger. Traction-force-activated payloads are bioinspired aptamer constructs attached to biomaterial scaffolds that selectively harvest, concentrate and reactivate multiple endogenous growth factors from cells, injury sites and blood lysate in vivo (rat femur and mouse skin) and ex vivo (human skin), at doses orders of magnitude lower than current clinical standards. Unmodified oligonucleotide aptamers retain functionality in enzyme-rich wound environments, substantially expanding the translational potential of nucleic-acid-based therapeutics. The ability to harvest and redeliver endogenous growth factors without exogenous triggers, recombinant proteins or cold-chain logistics via mechanoresponsive biomaterials opens possibilities for accessible, cost-effective combinatorial biologic therapies.

Postoperative surveillance for colorectal cancer (CRC) remains largely uniform despite potentially time-dependent recurrence hazards. Understanding temporal recurrence dynamics may help refine surveillance strategies. However, data on temporal recurrence patterns in CRC remain limited, particularly across stage, risk, and recurrence sites. This retrospective pooled individual participant data analysis integrated four RCTs conducted by the Japan Clinical Oncology Group. A total of 3655 patients with curatively resected pathological stage (pStage) II/III CRC were analysed (816 pStage II; 2839 pStage III). Time-dependent recurrence hazards were estimated using biweight kernel-smoothed hazard functions. Peak timing was defined as the maximum hazard within the first 5 years postoperatively. Analyses were stratified by pStage, prognostic factors identified using multivariable Cox regression, and recurrence site. Five-year relapse-free survival (RFS) was 87.9% in pStage II and 77.0% in pStage III. Peak recurrence hazard was observed at 10.0 and 12.8 months in pStage II and III, respectively. In pStage II, age &#x2265;&#x202f;70 years and pT4 were independent poor prognostic factors. In pStage III, age &#x2265;&#x202f;70 years, male sex, pT4, pN2 and absence of adjuvant chemotherapy (ACT) were independently associated with poorer RFS. Patients aged <&#x202f;70 years in pStage II showed earlier peak timing than those aged &#x2265;&#x202f;70 years (9.0 versus 12.9 months). In pStage III, patients without ACT showed earlier peak timing than those receiving ACT (3.3 versus 12.8 months). Site-specific analysis showed that liver metastasis had the earliest peak recurrence hazard (8.0 months), particularly in pStage II (6.0 months). Postoperative recurrence hazards showed distinct temporal patterns according to pStage, prognostic factors, and metastatic sites. These findings suggest that considering not only who is at risk, but also when the risk is highest, supports the development of risk- and time-adapted postoperative surveillance strategies.

Background: The latest revision of the McDonald criteria for diagnosis of multiple sclerosis (MS) establishes that the optic nerve can serve as a fifth anatomical location within the central nervous system for diagnosis. Optical coherence tomography (OCT) images can serve as evidence for this purpose. Objective: To assess the accuracy of automated artificial-intelligence-based classification of MS patients using OCT data obtained from two different centers. Methods: OCT data were collected from two centers using standardized APOSTEL-based protocols and similar equipment. Retinal layer thicknesses-mean and standard deviation (STD) values-were analyzed in four layers and in six regions per layer per eye. A support vector machine classifier with recursive feature elimination and Shapley additive explanations value analysis was applied to identify the most relevant features and maximize classification accuracy between control subject and MS patient eyes. Results: The database drawn from two hospitals comprised 112 eyes with MS without prior history of optic neuritis and 193 eyes of control subjects. The classifier achieved maximum accuracy (0.8459) using 20 input features. The mean and STD metrics had similar importance, with the most influential layers being the ganglion cell layer, inner plexiform layer, and the inner retinal layer complex. Key regions included the papillomacular bundle and the superior temporal perimacular area. Conclusions: OCT data facilitates highly accurate MS diagnosis across different centers. Artificial intelligence assessment could facilitate automated classification. These findings provide evidence of the important role of the optic nerve in MS diagnosis.

Children and adolescents with Attention-Deficit/Hyperactivity Disorder (ADHD) often show increased emotional dysregulation (ED) and internalizing emotional problems (iEP). However, the predictive and moderating role of these emotional difficulties in the treatment of ADHD-particularly regarding pharmacological (methylphenidate, MPH) and non-pharmacological approaches (neurofeedback at home, NF@home)-remains understudied. This study aims to evaluate the effectiveness of MPH and NF@home in reducing iEP/ED in children with ADHD and to assess the predictive role of iEP/ED and neuropsychological executive function (EF) measures in treatment outcomes. The per-protocol sample included 146 children (7-13&#x2009;years) diagnosed with ADHD (inattentive or combined type). iEP was measured with the Strengths and Difficulties Questionnaire (SDQ), ED via a dysregulation profile, and EF using the BRIEF and Continuous Performance Test (CPT). ADHD symptoms were assessed using the ADHD Rating Scale IV. Participants were randomized to either the MPH group (n&#x2009;=&#x2009;59) or the NF group (n&#x2009;=&#x2009;87), which included NFSMR (n&#x2009;=&#x2009;72) and NFTBR (n&#x2009;=&#x2009;15), based on individual Theta/Beta-Ratio (TBR). Mixed-model analyses considered symptom severity at baseline, age, gender, IQ, and study center as covariates. All intervention groups showed significant changes in iEP, but with different effects regarding parent (NFSMR: d&#x2009;=&#x2009;-0.396, 95% CI [-0.574, -0.217]; NFTBR: d&#x2009;=&#x2009;-0.367, 95% CI [-0.747, 0.013]; MPH: d&#x2009;=&#x2009;-0.409, 95% CI [-0.603, -0.215]) and teacher ratings (NFSMR: d&#x2009;=&#x2009;-0.211, 95% CI [-0.405, -0.017]; NFTBR: d&#x2009;=&#x2009;-0.639, 95% CI [-1.06, -0.222]; MPH: d&#x2009;=&#x2009;-0.142, 95% CI [-0.340, 0.056]). There was no between-intervention effect, supported by non-inferiority analyses. For ED, we also found significant effects for all interventions; however, significantly more pronounced effects were obtained for MPH. Moreover, both prediction models indicated a limited predictive value. The findings suggest non-inferiority of NF versus MPH treating iEP within an ADHD sample. However, due to limited predictive value, no clear clinical guidance can yet be drawn regarding personalized treatment recommendations. Further studies are needed to confirm and extend these findings.

Pilus-specific or class C sortases (SrtCs) catalyze pilus polymerization in Gram-positive bacteria and possess a critical lid near the active site that is in a closed or open conformation. The crystal structure of SrtC, which polymerizes endocarditis and biofilm-associated pili in Enterococcus faecalis, presented here at 1.9&#x2005;&#xc5; resolution (PDB entry 24rr), reveals the flexible lid in both conformations. Two molecules in the asymmetric unit exhibit different lid conformations stabilized by distinct sets of contacts. The DPX motif from the lid at the active site stabilizes the closed conformation, while the TPY motif within the conserved TP(Y/L)XIN(S/T)H motif of the &#x3b2;7-&#x3b2;8 loop from the asymmetric mate pointing towards the active site stabilizes the open conformation. For the first time, proline cis-trans isomerization, likely acting as a molecular switch in SrtC, was captured, regulating the access of secondary substrate to the active-site cleft by toggling between two states at the &#x3b2;7-&#x3b2;8 loop. Lid movement regulates primary-substrate (LPXTG sorting motif) binding and harmonizes conformational changes at the &#x3b2;7-&#x3b2;8 loop for secondary-substrate (YPKN pilin motif) binding. In silico analysis and RP-HPLC-based assays helped to map residues within sorting motifs near the scissile bond, providing insights into enzyme-substrate recognition. Removing the N-terminal lid region enhances E.&#xa0;faecalis SrtC activity. The unprecedented structural snapshots of both open and closed lid conformations, synchronized with the proline switch in the &#x3b2;7-&#x3b2;8 loop, captured in the same crystal structure, advance the understanding of sortase-mediated virulence in enterococci and provide a framework for the development of antivirulence strategies.

A comprehensive evaluation of allogeneic human bone-derived gelatin (hBG) was conducted as a promising material for regenerative medicine and 3D bioprinting. Comparative analysis with a commercial animal-derived gelatin product (hereinafter CDH) revealed a similar FTIR spectral profile, confirming the retention of key functional groups. Rheological analysis under steady shear showed that hBG is a pseudoplastic fluid with a concentration-dependent viscosity. However, a complete characterization of bioinks requires oscillatory measurements (G', G&#x2033;, and gelation point), which are planned in future studies. In vitro experiments on spheroid models (chondroblasts) demonstrated biocompatibility, an absence of cytotoxicity, and support for cell viability in 5% and 10% (hBG-5/10) matrices. Owing to its origin, the material allows for the modeling of "human-in-human" conditions, which makes it a promising precursor for the development of bioinks and matrices in regenerative medicine and tissue engineering. However, definitive positioning of the material as a bioink requires additional oscillatory rheological studies (determination of G', G&#x2033;, thixotropy, and gelation point), which were not performed in the present work.

Background/Objectives: Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive &#x3b2;-cell loss. Apolipoprotein CIII (apoCIII), a lipid metabolism regulator, is elevated in T1D and implicated in &#x3b2;-cell apoptosis. Antisense oligonucleotide-mediated apoCIII reduction delays diabetes onset in diabetes-prone BioBreeding (DPBB) rats. This study examined whether small-interfering RNA (siRNA) targeting apoCIII during the final prediabetic month preserves islet integrity and delays T1D onset. Methods: Two siRNAs targeting rat apoCIII were evaluated in 30-day-old DPBB rats for efficacy and off-target effects. Hepatic and plasma apoCIII levels were measured, and neighboring apolipoprotein gene expression was assessed. The most specific candidate (apoCIII-siRNA2) was selected. Duration of action was determined after a single injection. To study the effects of apoCIII-lowering treatment in vivo, islets from 25-day-old DPBB rats were transplanted into the anterior chamber of the eye of age-matched DPBB recipients. Rats received weekly intravenous injections of apoCIII-siRNA2 from day 30 until diabetes onset. Islet morphology, vascularization, and phagocyte infiltration were assessed by confocal imaging three and five weeks post-transplantation. Results: Both siRNAs reduced apoCIII, but one showed off-target effects and was excluded. A single injection of apoCIII-siRNA2 suppressed plasma apoCIII for approximately one week and weekly treatment maintained low circulating apoCIII levels. Five weeks after transplantation islet morphology and vascularization were preserved, and there was no increase in phagocyte infiltration. This resulted in a delayed onset of diabetes. Conclusions: siRNA-mediated apoCIII reduction delays pancreatic islet deterioration and T1D onset in DPBB rats, supporting apoCIII as a contributing factor to &#x3b2;-cell vulnerability and thereby a potential therapeutic target.

Ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T-cell (CAR-T) therapy, has transformed the management of relapsed or refractory multiple myeloma and demonstrated long-term, treatment-free remissions in patients (CARTITUDE-1: median overall survival, 60.7 months and 33% of patients with progression-free survival &#x2265;5 years). While pivotal clinical trials have demonstrated unprecedented depth and durability of response, the real-world populations can sometimes diverge from trial-eligible cohorts, with greater age variability, comorbidity burden, functional limitations, and logistic challenges that may impact outcomes. As cilta-cel adoption expands across treatment centers into the community and outpatient settings, generating real-world evidence and best practices is critical to ensuring that the transformative potential of this therapy is realized safely and effectively. This is a single-center report on our experience implementing cilta-cel. We share practical strategies that have supported favorable safety and clinical outcomes across the treatment continuum-beginning with patient selection, baseline risk assessment and pre-infusion optimization, through acute monitoring and management of adverse events, to longer-term follow-up. We highlight the importance of structured workflows, multidisciplinary coordination, early referral pathways, anticipatory guidance, and collaborative care models in achieving safe and effective outcomes. As centers continue to refine cilta-cel implementation, these institutional insights will play a key role in shaping best practices for patients receiving CAR-T therapy.

Background/Objectives: To evaluate long-term neuroretinal changes in patients with persistent COVID-19 (PC) versus asymptomatic controls, using optical coherence tomography (OCT) over a two-year follow-up. Methods: This prospective longitudinal study enrolled 133 participants: 94 PC patients and 39 control subjects. Participants underwent comprehensive ophthalmological assessment at baseline, at 1 year (voluntary), and at 2 years. Structural retinal changes were analysed using spectral-domain OCT with the Bruch's Membrane Opening-Minimum Rim Width and Posterior Pole protocols. Longitudinal analyses focused on within-subject change from baseline to 2 years and on between-group comparisons of these change scores, with Bonferroni correction applied within predefined families of comparisons. Results: The most robust corrected between-group longitudinal finding was a greater decrease in the central macular sector (C0) of the inner plexiform layer (IPL) in PC patients than in control subjects. Conclusions: Although these findings suggest possible subtle inner retinal involvement in PC patients, particularly in the central IPL, the results should be interpreted cautiously because of the modest control sample, control-group heterogeneity, and the exploratory nature of the sectoral OCT analyses. OCT may be useful in research settings as a complementary tool for exploring subtle neuroretinal changes in PC patients; however, its clinical utility and role as a validated biomarker remain unproven.

Serogroup X has emerged as an important cause of invasive meningococcal disease across Sub-Saharan Africa. Recent advances in serogroup X vaccines were reviewed. Early vaccine development efforts focused on outer membrane vesicle-based approaches. The growing epidemiological importance of serogroup X accelerated the development of serogroup X conjugate vaccines. Building on the success of MenAfriVac, PATH and the Serum Institute of India Pvt. Ltd. developed NmCV-5 (MenFive), the first pentavalent meningococcal conjugate vaccine targeting serogroup X. NmCV-5 achieved World Health Organization (WHO) pre-qualification in 2023, enabling deployment across Sub-Saharan Africa. A second pentavalent vaccine, EuNmCV-5, developed by Eubiologics is advancing through clinical development, while an additional candidate from Sinovac Biotech is entering early phase evaluation. These advances representative a major milestone in meningococcal disease prevention. Within the last fifteen years significant vaccine developments have been made to combat serogroup X. With NmCV-5 attaining WHO pre-qualification, strategies for its implementation within Sub-Saharan Africa are being devised. Only one vaccine is currently available in Sub- Saharan Africa, but trials of a second pentavalent ACWYX conjugate vaccine are ongoing. Carriage studies and disease surveillance are crucial to monitor serogroup X vaccine effectiveness.

The genetic dissection of complex traits in livestock continues to pose a significant challenge in the field of animal genetics and breeding. Although traditional genome-wide association studies (GWAS) are capable of localizing genetic variants associated with specific traits, they are insufficient to elucidate the underlying physiological mechanisms. An integrated analysis of multi-trait GWAS and multi-transcriptomic data systematically identifies key tissues and cell types influencing complex traits in beef cattle and elucidates their genetic regulatory basis. We systematically mapped tissue- and cell-type-specific regulatory architectures underlying 20 economically important traits in beef cattle. Tissue-level analyses revealed distinct trait-tissue associations: fatty acid traits, including C16:0 and C20:4, were enriched in liver; carcass traits, including marbling score and carcass weight, in renal cortex/medulla and longissimus dorsi muscle; meat-quality traits such as pH in cartilaginous tissues; and total fat content in bone marrow. At cellular resolution, analysis of eight trait-associated tissues identified 38 discrete cell types. Myofibers were significantly associated with most carcass traits, including rib-eye area and backfat thickness, whereas hepatocytes emerged as key regulators of fatty acid and meat-quality traits, such as C16:0 and crude protein content. Transcription factor analysis identified cell-type-specific regulators: TBX15, SOX6, and TCF12 in myofibers; FOXA2 and NR1H4 in hepatocytes; and IRF8 and IKZF1 in microglia. Notably, hepatocytes and microglia showed complementary, trait-specific association patterns: hepatocytes were enriched for C16:0 associated saturated fatty-acid metabolic pathways, while microglia were enriched for C16:1 and unsaturated fatty-acid-related pathways, suggesting potential cross-tissue coordination in lipid regulation. Our study links specific tissues and cell types to phenotypic variation in beef cattle and identifies core transcriptional regulators and pathways driving trait variation. These cell-resolved maps provide mechanistic insight into how genetic variation shapes economically important traits, offering a valuable resource for functional studies, cell-informed precision breeding strategies, and the design of large-scale molecular phenotyping.

In recent years, activation of the immune system has been recognized as a key mechanism in supporting the therapeutic outcome of oncolytic virotherapy for cancer. Most studies have focused on T cells in the characterization of immune responses to therapy; however, the role of dendritic cells (DCs) in priming these cytotoxic T cell responses has been far less explored. rVSV-NDV is a chimeric oncolytic virus that causes a highly immunogenic cell death in infected tumor cells. In tumor-bearing mice, this leads to increased levels of circulating and tumor-infiltrating tumor-specific CD8+ T cells upon rVSV-NDV treatment. Here, we show that the rVSV-NDV-mediated immunogenic cell death leads to the activation of the dendritic cell subtypes, cDC1 and cDC2, in vitro and in vivo. Using wild-type (WT) and cDC1-deficient (BATF3KO) mice implanted with B16 melanoma lesions, we observed that the increase in circulatory, tumor-specific CD8+ T cells and the survival benefit conferred by rVSV-NDV tumor therapy depends on a functional cDC1 population. Finally, we demonstrate that rVSV-NDV therapy can be significantly augmented by the combined treatment with adoptively transferred cDC1 cells that were ex vivo activated and antigen-loaded by co-culture with rVSV-NDV-infected tumor cells.

The urethra, lined by epithelial cells, serves as the conduit for urine outflow from the body. The epithelial lining of the urethra comprises distinct cell types with gene signatures indicative of roles in antimicrobial and immune defense. Resident macrophages embedded in the urethral epithelial layer represent a transcriptionally distinct subtype with purported roles in immune surveillance and antigen presentation. Urinary pathogens ascend up the urethra to reach the bladder. A study of the urethral epithelial lining and associated immune cells will shed light on host defense mechanisms in the lower urinary tract. A central goal of this protocol is to provide optimized methods for dissecting the female mouse urethra and isolating the epithelial lining for downstream analyses. The protocol describes an optimized method for isolating epithelial tissue from the female mouse urethra by gentle enzymatic and mechanical separation. Subsequent to epithelial isolation, methods for gentle enzymatic digestion of isolated urethral epithelium and associated immune cells to obtain high-viability single-cell suspensions are described. The protocol also details methods for flow cytometry analysis of the isolated mouse urethral epithelial cells and epithelial-associated immune cells. Additionally, methods to generate stratified 3D urethral epithelial organoids from isolated epithelial cells are presented here. The protocol also details an optimized method for whole-mount immunostaining of urethral epithelial sheets, which can be used to observe the morphology and molecular structures of mouse urethral epithelium and associated immune cells. Overall, the methods described here for isolation and single-cell digestion of mouse urethral epithelial cells enable downstream analyses, including immunostaining, flow cytometry, organoid generation, and single-cell RNA-sequencing.

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Cancer immunotherapy represents a paradigm shift in oncology. Immunotherapeutic approaches are approved in a growing number of clinical indications, spanning acute leukemias to cervical cancer. Response rates vary substantially, both within and across diseases and range from limited responses to cures. At the same time, the underlying drugs are diversifying and so are their modes of action. Immunotherapies now also transcend the boundaries of oncology and are under active investigation in, amongst other diseases, autoimmunity. Here, early clinical trials are suggesting relevant efficacy. In this context, a thorough understanding of these principles underlying currently approved immunotherapies, is more important than ever, to guide clinicians in their patient management and care. The present work gives an overview of the modes of actions of current cancer immunotherapies and gives a resume of current problems and challenges.

Oral submucous fibrosis (OSF) is a chronic, potentially malignant disorder strongly associated with areca nut chewing. Cuproptosis, a newly identified form of cell death, may play a role in OSF progression, but the mechanisms remain unclear. This study aimed to explore the involvement of cuproptosis in OSF. Here, we observed a significant increase in cuproptosis markers (FDX1 and LIAS) in OSF lesion tissues compared to normal oral mucosa. In vitro, arecoline induced cuproptosis predominantly in epithelial cells, characterized by increased FDX1 and LIAS expression, disrupted lipoylation of DLAT, and elevated intracellular Cu&#xb2;&#x207a; accumulation. An epigenetic modification PCR array revealed that arecoline most markedly upregulated PUS1, a pseudouridine (&#x3a8;) modification synthase. Functional knockdown of PUS1 alleviated arecoline-induced cuproptosis, as evidenced by reduced Cu&#xb2;&#x207a; levels and restored lipoic acid expression. Dot blot and PA-&#x3c8;-seq further demonstrated that arecoline-induced PUS1 upregulation altered the global tRNA pseudouridylation landscape, with enrichment of &#x3a8; peaks in Wnt- and copper transport-related transcripts. PUS1 knockdown markedly attenuated arecoline-induced &#x3b2;-catenin nuclear localization and the upregulation of MYC, FZD6, CTNNB1, NFAT5, SLC25A39, and VDAC2. In conclusion, we reveal a previously unrecognized mechanism whereby arecoline promotes OSF via PUS1-dependent pseudouridylation remodeling and epithelial cuproptosis, offering novel therapeutic targets for OSF intervention. The online version contains supplementary material available at 10.1007/s13205-026-04931-8.