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Influenza causes 650,000 deaths, 3-5 million hospitalizations, and 1 billion cases worldwide each year. There is a limited repertoire of antivirals available to tackle this burden, highlighting the risk of developing resistance to current drugs. An effort to develop new influenza antivirals has been made, leading to the approval of baloxavir. Some mutations leading to reduced susceptibility to baloxavir have been reported, but information about their epidemiology is scarce. Thus, we aim to evaluate the prevalence of substitutions in the polymerase acidic (PA) subunit associated with baloxavir susceptibility over 16 epidemiological seasons in Europe. We evaluated 87,266 sequences collected from 2009 to 2025 in Europe, annotated the mutations, and identified all known amino acid substitutions related to changes in baloxavir susceptibility to assess their prevalence. A total of 149 (0.2%) sequences with at least one substitution were identified, 81 (0.09%) exhibiting reduced susceptibility. Overall, 17 different substitutions were detected, located both inside and outside baloxavir binding site. The number of substitutions detected ranged from 0 to 15 per season, with E23K, E23R, K34R, A36V, I38F, I38L, and E120D emerging after baloxavir approval in Europe. Double and triple concurrent substitutions were also identified. While the prevalence of sequences with substitutions that alter baloxavir susceptibility remains stable, the number of circulating substitutions increases over time. This implies the emergence of amino acid substitutions that did not circulate before and the concurrence of double and triple substitutions that might synergize their individual effects. These results highlight the need for virological surveillance and novel antiviral treatments.

The genus Sesamum (Pedaliaceae) comprises a wide range of cultivated and wild species. Sesame (Sesamum indicum) is recognized as one of the oldest oilseed crops cultivated worldwide, whereas S. schinzianum is a wild relative closely associated with the evolutionary history of cultivated sesame. Although the nuclear and chloroplast genomes of Sesamum species have been investigated in previous studies, mitochondrial genome evolution within the genus has received relatively limited attention. In this study, we assembled and comparatively analyzed the mitochondrial genomes of S. indicum and S. schinzianum by integrating BGI short-read sequencing and Oxford Nanopore long-read sequencing data. Genome assembly was performed using Flye and Unicycler, and annotation was conducted using PMGA together with manual curation. Comparative analyses were then carried out to examine genome organization, gene content, repetitive sequences, codon usage, RNA editing sites, chloroplast-derived sequences, phylogenetic relationships, and collinearity patterns. The mitochondrial genomes of S. indicum and S. schinzianum were assembled into one circular molecule and two major circular contigs, respectively, and both contained 36 conserved protein-coding genes. Abundant simple sequence repeats dominated by tetranucleotide motifs and notable repeat variation were detected. Codon usage showed moderate bias, and 478 and 455 RNA editing sites were predicted in S. indicum and S. schinzianum, respectively. Chloroplast-derived sequences accounted for 8.50% and 6.96% of the mitochondrial genomes, respectively. Phylogenetic and collinearity analyses supported a close relationship between the two Sesamum species and identified synteny-based structural differences between their mitogenomes. Comparative analysis of mitochondrial- and chloroplast-based phylogenies showed that the two datasets were largely congruent at the family level and consistently supported the close relationship between the two Sesamum species, although they differed in the placement of several deeper lineages. These results suggest that Sesamum mitogenomes retain conserved gene content while showing assembly- and synteny-supported structural differences. This study provides useful genomic resources for comparative and evolutionary studies of mitochondrial genomes in Pedaliaceae.

The ability to access atomically tailored complex peptides and proteins provides powerful opportunities for dissecting molecular functions and advancing applications in chemical biology, therapeutics, and (bio)-materials science. Robust precision-engineering strategies are essential to construct well-defined protein architectures while preserving native folding and activity. To do so, chemoselective bioconjugation techniques have been developed to modify specific side chains of amino acids. This allowed for the selective introduction of functionalities on predetermined amino acids. However, ultimate control can be achieved only through site-selective modifications that precisely define both the nature of the linkage and the exact position of conjugation on elongated peptide sequences or fully assembled proteins. Cysteine residues are of particular interest, as their highly nucleophilic thiols offer excellent chemoselectivity and typically occur in low abundance in their reduced form. Here, we examine chemoselective transformations targeting cysteine residues that have been further refined to occur exclusively at predefined positions within a peptide or protein, thereby achieving a high degree of site-selectivity. This review focuses exclusively on chemical strategies for cysteine modification, offering guidance for future synthetic developments within the field of precision chemistry. Achieving this level of precision requires advanced chemical strategies that exploit the local environment of the targeted cysteine. One approach involves leveraging neighboring functional groups, for example, engaging the thiol together with the α-amine or carboxylate to enable selective N- or C-terminal modification, respectively. In such designs, the cysteine side chain may contribute through transient interactions, direct incorporation into the covalent linkage, or the stabilization of the desired product. Recently, a promising strategy has attracted increasing attention in which site-selectivity is enabled by temporary interaction with a proximal amine, thus being applicable to differentiate also between internal cysteines. Together, these strategies highlight that site-selective protein modification has evolved into a powerful tool for the rational design and functional control of complex biomolecules, redefining what is achievable in chemical biology, therapeutics, and biomaterials science. We anticipate that increasingly routine or user-friendly approaches such as the programmable TriTEx method will further accelerate the adoption of precision biomolecule conjugates in both research and industrial settings.

Four species of tardigrades have been found in the meiofauna communities of the seagrass meadows and adjacent areas off the coast of Vietnam. For two of them, Batillipes binhdinhicus sp. nov. and Batillipes ampullus sp. nov., integrative descriptions are presented. Batillipes binhdinhicus sp. nov. belongs to the B1 subgroup of species by having toe 3 shorter than toe 4 on legs IV, while Batillipes ampullus sp. nov. with toes 3 and 4 on legs IV of equal length belongs to the A group of species. Batillipes binhdinhicus sp. nov. can be distinguished from all previously described species by a set of features: very long cephalic cirri and leg sensory spines, both with swollen tips, presence of a single pair of conical lateral body projections between all pairs of legs and simple triangular caudal appendage. The distinctive features for Batillipes ampullus sp. nov. are short sensory organs on the fourth pair of legs, which consists of two parts and have swollen tips with a tuft of additional sensory filaments that are structurally similar to those found on the cephalic appendages. In addition, Batillipes ampullus sp. nov. does not have noticeable lateral body projections between the legs, and the body ends in a semi-circular tail appendage. Based on the obtained data on the nucleotide sequences of 18S rDNA and 28S rDNA, the distinction between the new species was confirmed.

Dipolar recoupling techniques play a fundamental role in solid-state NMR spectroscopy, enabling precise structural characterization of solid materials under magic-angle spinning (MAS). Among these, the supercycled R41 2 (SR4) stands out for its efficiency and stability in recoupling heteronuclear dipolar interactions under fast MAS conditions (&#x2265;40 kHz). However, its performance significantly degrades at slow-to-moderate MAS rates (<35 kHz), which restricts its wide applications. To overcome this limitation, herein we introduce a practical modification of SR4 through the strategic incorporation of adjustable window delays between the recoupling &#x3c0; pulses, dubbed windowed SR4 (wSR4). This design introduces the variability of pulse duration and rf amplitude, which simplifies the recoupling optimization procedure, while enabling enhanced recoupling performance and improved tolerance to experimental imperfections. Theoretical analysis and NMR experiments collectively confirm that wSR4 substantially boosts heteronuclear dipolar recoupling efficiency while effectively suppressing undesired spin interactions, particularly under slow MAS conditions. The practical utility of wSR4 is further validated in in situ NMR experiments on SAPO zeolite, where the markedly improved 2D correlation spectroscopy provides deeper insights into local structures. This approach enables precise dipolar measurement, efficient polarization transfer, and the establishment of high-fidelity heteronuclear correlations, all of which are critical for structural elucidation in complex solid systems. Moreover, the windowed SR4 design strategy introduced here is expected to provide a generalizable framework for optimizing symmetry-based recoupling sequences across a wide range of MAS conditions.

CD1d-restricted invariant natural killer (iNK) T cells are innate T cells known for their ability to shape adaptive immunity toward inflammation or immune-suppression via the rapid production of Th1-, Th2-, and Th17-type cytokines from corresponding iNKT subsets such as NKT1, NKT2, and NKT17. IL-10-producing invariant NKT cells, termed NKT10 cells, are thought to play an immunoregulatory role, but their potential clinical use remains underexplored. We characterized human NKT10 cells from cord-derived iNKT cells and investigated their therapeutic utility in allogeneic stem cell transplantation. Cord and cord-derived iNKT cells contained a high frequency of CD4+CD25+CD161lowFoxP3+ iNKT cells and showed Th2/Th10-biased cytokine production upon antigenic stimulation. Accordingly, cord-derived iNKT cells displayed a distinct gene expression profile with upregulated genes related to NKT2, NKT10, and regulatory T cells compared with adult donor-derived iNKT cells. Furthermore, single-cell RNA sequencing analysis of cord-derived iNKT cells confirmed the presence of NKT10-like subset that was enriched with multiple immunoregulatory pathways and genes related to immune-checkpoints (NRP1, PD1, CLTA-4, and GITR) and NKT10 (MAF, HIF1A, and FoxP3), whereas the NKT1/17-like subset present in adult donor-derived iNKT cells showed upregulation of genes related to cytotoxicity (GZMA/B, KLRD1, and PRF1), NKR (KLRK1, KLRB1, KLRG1, and NKG7), NKT1 (EOMES and TBX21), and NKT17 (RORC). Lastly, cord-derived iNKT cells suppressed alloreactive T cell proliferation in vitro and ameliorated xenogeneic graft-versus-host disease where the immunodeficient NSG mice received human peripheral blood mononuclear cells supplemented with cord-derived iNKT cells. Thus, NKT10-enriched, cord-derived iNKT cells are candidate cell therapeutics for immune-modulation in allogeneic stem cell transplantation and other autoimmune diseases.

Photoautotrophic micropropagation (PAM), which employs sugar-free media and ventilated culture vessels to enhance plantlet photoautotrophic capacity and overall quality, has substantial potential to increase propagation efficiency when integrated with an appropriate propagation protocol. In this study, we established a PAM propagation protocol for medicinal cannabis (Cannabis sativa L.) using two genotypically different cultivars, 'Charlotte' and 'Auto Charlotte', and systematically evaluated the genetic similarity of plantlets across all successive batches via inter-simple sequence repeat (ISSR) markers as well as their performance after transplanting. Furthermore, a cultivar-specific logistic growth model was used to estimate annual yield and optimize the mother plant culture cycle for maximizing production. Results showed that under PAM conditions, mother plants sustained 11 batches of shoot tip harvest within a 98-d cycle. The propagation coefficients of 'Charlotte' and 'Auto Charlotte' reached 12.0 (95%CI: 11.0-13.0) and 11.3 (95%CI: 10.5-12.2), respectively. After 35 days of culture, plantlets from all batches exhibited uniform morphological and physiological traits, with rooting rates exceeding 70%. These plantlets could be directly transplanted without acclimatization, and minor growth differences between batches were eliminated by extending the culture cycle by one week. ISSR markers detected no obvious polymorphism between regenerated plantlets and their respective mother plants, with all similarity coefficients exceeding 0.90. Model simulations suggested that a 70-day culture cycle could maximize annual production, reaching a yield of 50,417-54,034 plants m-2. In conclusion, this study established an efficient PAM propagation protocol with high genetic similarity for the two tested medicinal cannabis cultivars, thereby providing technical support for cannabis plantlet production.

Human gut microbial communities capable of degrading mucin are taxonomically unique and have a range of physiologically relevant metabolic outputs. To determine the feasibility of reviving mucin-degrading fecal microbial communities after cryopreservation, we employed 16S rRNA gene sequencing to characterize revived communities. Microbial communities were generally stable but small donor-dependent shifts in diversity, composition, and taxonomy were observed following revival. The revivability of these microbial communities is valuable for studying mucin-degrading microbial communities.

Guillain-Barr&#xe9; syndrome (GBS) is a rare, immune-mediated inflammatory disease of the complex peripheral nervous system that often follows acute infections, and may also be associated with long-term 'silent infections'. Long-term "silent infections" can alter the gut microbiota, which in turn may contribute to immune-mediated inflammatory diseases. Emerging evidence suggests that gut dysbiosis and altered serum metabolites are associated with GBS, but the causative link between GBS and gut microbiota remains unclear. Therefore, this study aimed to evaluate the association between gut microbiota structure and serum metabolic profile in GBS. Untargeted metabolomics profiling of serum and metagenomics sequencing of stool samples were performed to capture the global metabolic and microbial differences between GBS subjects and healthy controls. Multivariate statistical analyses, including PLS-DA, were applied to identify distinct clustering patterns and differential abundances of metabolites and gut microbiota. Pearson's correlation analysis was used to estimate the correlations between abundance of gut microbiota and serum metabolic profile. Seven different media were used to isolate the potential pathogens from GBS stool samples. The metabolome data revealed that gamma-aminobutyric acid (GABA) metabolism and secondary cholic acid metabolism were perturbed in GBS. Specifically, GABA was increased significantly (approximately 14.3-fold), while multiple secondary cholic acids (methyl deoxycholate, glycodeoxycholic acid, glycolithocholic acid, taurolithocholic acid, and coprocholic acid) were decreased significantly in GBS subjects. Regarding the gut microbiota identified via metagenomic sequencing of stool samples, Ligilactobacillus salivarius, Enterocloster bolteae, and the opportunistic pathogenic Klebsiella pneumonia were notably more abundant in GBS subjects, while Bacteroides sp., Roseburia hominis and Paraprevotella xylaniphila were decreased significantly. In addition, pathogens such as K. pneumoniae were also isolated from GBS subjects. Further analysis of the metagenomic data revealed enrichment of prokaryotic genes involved in the GABA biosynthesis pathway, while genes associated with secondary cholic acid metabolism pathways were decreased in gut microbiome in GBS subjects. On this basis, correlation analysis revealed that changes in GABA were associated with altered levels of gut microbes including Enterococcus species, Ligilactobacillus salivarius and Enterocloster bolteae, whereas changes in secondary cholic acids were positively correlated with altered levels of Bacteroides species and Roseburia species. GABA metabolism and secondary cholic acid metabolism were significantly disturbed in GBS subjects, potentially resulting from the dysbiosis of the gut microbiota. K. pneumonia and other no gut microbes were significantly enriched and isolated in GBS and may contribute to the inflammatory response in this immune-mediated inflammatory disease. These findings also suggest that GABA may be a promising biomarker for the diagnosis of GBS and that modulation of gut microbiota might impact the clinical course of GBS.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a high risk of relapse. Leukemia cutis is characterized by the presence of neoplastic leukocytes infiltrating the skin. Here, we present a previously treated AML patient who relapsed with isolated leukemia cutis without bone marrow involvement. Fluorescence in situ hybridization (FISH) and RNA sequencing of the skin lesion revealed a KMT2A::MLLT6 rearrangement, which enabled the use of revumenib. This case highlights the diagnostic complexity and therapeutic implications of isolated extramedullary AML relapse and underscores the need to perform genetic testing at the site of disease recurrence-especially without bone marrow involvement of relapsed disease. The authors have confirmed clinical trial registration is not needed for this submission.

Differences in enhancer activity between species can help drive phenotypic diversity, yet enhancers often have conserved functions despite rapid sequence evolution, posing a challenge for quantifying their functional differences between species. Previous machine learning models have focused on the binary task of predicting differences in the presence of enhancers between species but have yet to demonstrate an ability to predict continuous differences in enhancer activity. Here, we trained convolutional neural networks on a regression task to predict chromatin accessibility-a proxy for enhancer activity-in the liver across five mammals, and we developed a novel framework to evaluate cross-species performance. We demonstrated that training on multiple species improves model generalization to both species used in training and held-out species. However, the models consistently achieved poor performance in predicting quantitative differences in accessibility between species at orthologous regions. Our study highlights the challenges in using regression models to predict chromatin accessibility changes between species. All data and code are available at&#xa0;http://daphne.compbio.cs.cmu.edu/files/azstephe/liver_regression_resource/ and https://figshare.com/projects/liverRegression/274293.

Biallelic loss-of-function mutations in the GTPase of immunity-associated protein 5 (GIMAP5) cause a severe syndrome characterized by altered immunity, lymphoproliferation, and progressive hepatopathy. This study aims to delineate the immunophenotypic signatures and clinical management of this deficiency by reporting the first Italian pediatric case alongside his mono-allelic carrier twin, integrated with a review of twenty previously published patients. We utilized trio-based clinical exome sequencing, flow cytometric immunophenotyping, protein expression profiling, and curated database analysis to evaluate the clinical trajectories. The 11-year-old proband presented with autoimmune cytopenias, severe viral infections, and early biochemical liver anomalies. Genetic analysis identified compound heterozygous variants (p.Leu204Pro and p.Arg214Ter) in GIMAP5 gene resulting in absent protein expression, alongside an expanded atypical memory B-cell population and T-cell exhaustion. Notably, his dizygotic twin harbored the heterozygous p.Leu204Pro variant and exhibited decreased protein expression coupled with a localized fibro-adipose vascular anomaly, but lacked immune defects. This observation suggests complex genotype-phenotype interactions, raising the question of whether partial GIMAP5 deficiency might subtly predispose to localized vascular anomalies under specific environmental or epigenetic conditions, likely requiring multi-hit mechanisms. Therapeutically, the proband achieved sustained clinical remission of cytopenias through immunomodulation with the mTOR inhibitor sirolimus. These findings expand the phenotypic spectrum of this disorder, underscoring a dual role in immune and endothelial homeostasis. Furthermore, the successful application of sirolimus highlights the efficacy of precision medical management as a viable strategy to stabilize patients, allowing time to carefully weigh the risks and benefits of definitive interventions, such as hematopoietic stem cell transplantation.

Drought stress presents a major challenge to global agriculture. FCS-Like Zinc Finger (FLZ) genes are transcriptionally responsive to environmental stimuli, yet their precise biological functions remain insufficiently characterized. Here, we demonstrate that OsFLZ5, a drought- and abscisic acid (ABA)-induced gene, enhances drought tolerance and ABA sensitivity in rice (Oryza sativa L.), but does not exhibit obvious adverse effects on crucial agronomic traits including yield and plant height. Overexpression (OE) of OsFLZ5 significantly improved survival rate, seedling growth, and biomass under both polyethylene glycol6000 (PEG6000)-simulated and soil-based drought conditions, whereas knockout (KO) lines exhibited impaired performance. Consistently, OE lines showed heightened sensitivity to ABA, while KO mutants were less responsive. OsFLZ5 promotes soluble sugar accumulation and reduces malondialdehyde and hydrogen peroxide levels after drought exposure. RNA sequencing revealed that OsFLZ5 modulates a suite of drought-responsive genes. Notably, we identified the bZIP transcription factor OsbZIP23 as a direct upstream regulator of OsFLZ5; it binds to the OsFLZ5 promoter and activates its expression, thereby contributing to enhanced drought resilience and ABA sensitivity. These findings establish the OsbZIP23-OsFLZ5 regulatory module as a new mechanism in the rice drought and ABA response, providing targets for developing drought-tolerant crops.

Turner Syndrome (TS), caused by complete or partial monosomy of chromosome X, is associated with increased morbidity and reduced life expectancy, largely driven by cardiovascular, endocrine, and metabolic complications. Here, we report the case of a 75-year-old woman with a confirmed diagnosis of non-mosaic TS (45,X) who remains clinically stable and functionally independent, beyond the expected survival for this condition. Notably, her mother is currently 101 years old, indicating a familial background of exceptional longevity. To explore the potential contribution of inherited genetic factors to this favorable phenotype, whole-genome sequencing (WGS) was performed in both the TS patient and her centenarian mother aiming to identify variants potentially associated with healthy aging and longevity. Hematological and biochemical analyses were subsequently used to functionally support the genetic and clinical findings, revealing preserved metabolic, inflammatory, and hematological profiles for the patient age. Together, these data suggest that a favorable genetic background may partially counterbalance the risks traditionally associated with X chromosome monosomy, contributing to preserved functional status into older age.

Congenital cataract is an important cause of childhood visual impairment, and genetic factors contribute substantially to its etiology. We report a boy aged 4-5 years with congenital cataract from a family with an apparent autosomal dominant inheritance pattern. Comprehensive ophthalmic examinations were performed in the proband and available relatives. Whole-exome sequencing (WES) in the proband identified several candidate variants, including a heterozygous CRYGD c.391T>C (p.Trp131Arg) variant. Subsequent Sanger sequencing was used to validate the candidate variants and assess segregation in available family members. The CRYGD c.391T>C (p.Trp131Arg) variant was present in all tested affected family members and absent in the tested unaffected relative, supporting co-segregation with the disease phenotype in this pedigree. Additional variants identified by WES, including ITM2B c.537C>G (p.Asn179Lys) and ASB10 c.1402T>C (p.Ter468GlnextTer6), showed segregation patterns that were less consistent with the familial phenotype. According to ACMG criteria, the CRYGD variant remained classified as a variant of uncertain significance. To provide limited biological context, we also reviewed a public gene expression dataset and found lens-enriched expression of CRYGD, consistent with its established role in lens biology. This case may provide useful evidence for future variant interpretation and genetic counseling in congenital cataract families carrying CRYGD variants; however, further functional studies are required to clarify the pathogenic significance of c.391T>C (p.Trp131Arg).

The raccoon (Procyon lotor), an invasive species in Europe, is a potential reservoir for zoonotic pathogens, posing risks to human, animal, and environmental health. This study investigates the prevalence and genetic diversity of Giardia duodenalis and Cryptosporidium spp. in raccoons from the Upper Lusatian Heath and Pond Landscape (Saxony, Germany), adopting a One Health perspective at the wildlife-aquaculture interface. Fecal samples (n&#xa0;=&#xa0;104) were collected from culled raccoons (2020-2022) across six pond farming areas. Samples were screened using rapid immunochromatographic antigen tests, followed by molecular characterization via PCR and sequencing of specific loci (gdh, tpi, and bg for Giardia; SSU rRNA and gp60 for Cryptosporidium). Giardia duodenalis assemblage B (sub-assemblage BIV) was detected in 24% of samples. Significant associations were observed with location (p&#xa0;=&#xa0;0.038) and aquaculture company (p&#xa0;=&#xa0;0.028). Cryptosporidium sp. skunk genotype was identified in 2% of samples, showing no significant correlation with the tested parameters. The identification of G. duodenalis (BIV) and Cryptosporidium skunk genotype highlights the role of raccoons as potential reservoirs in water-rich production landscapes. The high prevalence of Giardia and its spatial heterogeneity suggest that site-specific pond management and raccoon dynamics might influence pathogen loads. This study provides the first molecular evidence of zoonotic protozoa in raccoons within this specialized aquaculture context. The results underscore the necessity of integrating wildlife monitoring into aquaculture management. Aligning invasive species control with One Health surveillance is essential to mitigate zoonotic risks and safeguard public health in aquatic ecosystems.

Caeconyx papso sp. nov. within the family Uristidae is described from the Porcupine Abyssal Plain, Northeast Atlantic Ocean. The new species differs from the only other member of the genus, Caeconyx caeculus, in possessing a triangular, sub-acute eye lobe, a convex and broadly rounded posterodistal margin of epimeron 1, and propodi of pereopods 3 and 4 that are not slender or elongate. Molecular sequence data for COI, H3, 16S, and 28S genes are provided for C. papso sp. nov. Phylogenetic analyses were conducted using this sequence data to assess the relationships between the new species, other members of the family Uristidae, and morphologically allied taxa within the family Tryphosidae. The new species is fully illustrated, and a key to species within Caeconyx is presented.

Backgrounds: Tertiary lymphoid structures (TLSs) are increasingly recognized as modulators of anti-tumor immunity, yet their clinical relevance in bladder cancer remains incompletely understood, partly owing to heterogeneity in their maturation states. Here, we demonstrate that germinal center (GC)-like TLS maturity, rather than TLS presence alone, is closely associated with immune activation and therapeutic response to Programmed Death-Ligand 1 (PD-L1) blockade in bladder cancer. The objective of this study was to systematically investigate the clinical significance, biological function, and therapeutic potential of tertiary lymphoid structure (TLS) maturation in bladder cancer. Specifically, we aimed to determine whether GC-like TLS maturity provides prognostic and predictive value beyond TLS presence alone, to elucidate the immune programs and tumor microenvironment remodeling associated with TLS maturation, and to explore whether TLS maturation can be therapeutically induced to enhance responsiveness to PD-L1 blockade. Methods: We performed an integrative analysis combining multi-cohort transcriptomics, spatially resolved histopathology, single-cell RNA sequencing, and functional murine experiments. TLS maturation states were defined using gene-expression-based GC-like TLS signatures and validated through multiplex immunohistochemistry. Clinical relevance was assessed in public immunotherapy cohorts and an independent neoadjuvant PD-L1-treated muscle-invasive bladder cancer (MIBC) cohort. Tumor immune microenvironment remodeling and chemokine-mediated cellular crosstalk were analyzed using deconvolution, Weighted Gene Co-expression Network Analysis (WGCNA), and CellChat. The therapeutic inducibility of TLS maturation was examined using a lymphotoxin-&#x3b2; receptor (LT&#x3b2;R) agonist in combination with PD-L1 blockade in a syngeneic bladder cancer model. Results: Across multiple transcriptomic cohorts, tumors enriched for GC-like TLS signatures exhibited significantly prolonged survival and higher objective response rates to anti-PD-L1 therapy, whereas less mature TLS phenotypes showed no consistent association with clinical association. These observations were independently validated in a neoadjuvant PD-L1-treated muscle-invasive bladder cancer cohort, in which high mature TLS density was associated with major pathological response and prolonged event-free survival, outperforming PD-L1 expression. Integrative histopathological and transcriptomic analyses indicated that GC formation marks a functional transition linking humoral immune programs with cytotoxic effector activity and shaping a memory-prone, pro-inflammatory tumor immune microenvironment. Chemokine signaling via the CC chemokine ligand 21 (CCL21)-C-C chemokine receptor type 7 (CCR7) and C-X-C motif chemokine ligand 12 (CXCL12)-C-X-C chemokine receptor type 4 (CXCR4) axes was strongly associated with TLS maturation and spatial organization. Finally, in a syngeneic bladder cancer model, pharmacological activation of lymphotoxin-&#x3b2; receptor signaling promoted TLS maturation and enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Together, these findings suggest that GC-like TLS maturity represents a clinically relevant biomarker and a potential therapeutic entry point for precision immunotherapy in bladder cancer. Therapeutic strategies that promote TLS maturation may convert immune-cold tumors into checkpoint-responsive states, providing a mechanistically grounded precision immunotherapy approach.

Muscle-specific tyrosine kinase antibody-positive myasthenia gravis (MuSK-MG), a subtype of myasthenia gravis (MG), follows an acute and severe disease course with suboptimal responses to standard treatments. Ofatumumab is a fully humanized anti-CD20 monoclonal antibody that induces potent and sustained B-cell depletion. Nonetheless, there are few reports on its use in sequential therapy failure after the failure of conventional drug treatment in MuSK-MG patients, especially in the Asian population. We report a 32-year-old female patient with anti-MuSK antibody-positive myasthenia gravis who was treated with ofatumumab as a rescue therapy after failing multiple treatments such as intravenous immunoglobulin (IVIG), plasma exchange, and efgartigimod, progressed to a crisis state necessitating prolonged mechanical ventilation. After the addition of ofatumumab to the combination of tacrolimus and corticosteroids, the clinical symptoms were significantly improved, the patient was successfully weaned from ventilator-assisted ventilation, the bulbar function was improved, and the limb muscle strength was significantly restored, and the patient was finally discharged uneventfully. This case is the first report of ofatumumab successfully treating refractory MuSK-MG in China, providing real-world evidence for its pivotal role in the treatment sequence. It also suggests that its characteristics, such as subcutaneous administration and low immunogenicity, may render it particularly suitable for high-risk patients, including those with concurrent infections.