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The LytR-CpsA-Psr (Lcp) family of proteins are highly abundant amongst actinomycetes and Gram-positive bacteria, and known to exist as multiple paralogs in an organism. Several reports demonstrated the essential LcpA to play a crucial role in coupling cell wall teichoic acids (arabinogalactan in mycobacteria) to peptidoglycan, making it an attractive drug target against tuberculosis (TB). However, apart from LcpB that accommodates capsular biosynthesis, role of the non-essential Lcp paralogs in mycobacteria largely remains elusive. Therefore, we made a comprehensive approach in characterizing the role of these non-essential Lcp proteins in mycobacterial cell wall assembly. Additionally, we used homology and interactive models as an alternate approach to X-ray crystallography, to identify hotspot residues for Lcp inhibition and subsequent bactericidal activity. Single and double knockout strains of each of the three Mycolicibacterium smegmatis (M. smegmatis) non-essential lcp genes were generated, out of which ΔlcpBΔlcpC exhibited severely compromised cell envelope with impeding cell division, altered morphology, reduced biofilms, enhanced susceptibility to stress, loss of capsular ⍺-glucans and exposed Galf residues. The higher pyrophosphatase activity of LcpB and LcpC, their interaction in native conditions and reduced AG/PG markers in ΔlcpBΔlcpC, substantiates their role in AG-PG ligation. Higher expression of lcpB/lcpC irrespective of external stress indicates tight regulation and coping mechanism conferred by these two homologs on the mycobacterial cell. Cell wall targeting anti-TB drugs in combination with two Lcp inhibitory compounds, Entrectinib and Sorafenib, proved bactericidal. This study highlights the primary role of LcpB/LcpC alongside LcpA in maintaining structural integrity of the M. smegmatis cell envelope, and combined anti-Lcp drug strategy as a novel approach to inhibit mycobacterial replication. The online version contains supplementary material available at 10.1186/s12866-026-04722-4.
Diagnostic advancements and classification updates appear to have reshaped the natural history of smoldering multiple myeloma (SMM), though this evolution has not been empirically quantified. We conducted a systematic review to evaluate temporal changes in SMM prognosis. PubMed, EMBASE and the Cochrane library databases were searched from January 1990 to November 2025, yielding 1415 reports, of which 14 studies met inclusion criteria. To reconstruct individual-level data, published time to progression (TTP) curves were digitized using a Shiny web application. Kaplan-Meier (KM) survival curves and meta-analyses were generated in RStudio. PROSPERO ID 1068697. Progression risk at two and ten years for all-risk patients was 22.8% and 60.1%, respectively; these increased to 44.7% and 85.6% in high-risk patients. Landmark analysis from year five revealed attenuated progression rates: 14.2% and 30.8% for all-risk, and 22.5% and 50.6% for high-risk patients at two and five years post-landmark, respectively. Studies enrolling most patients before 2014 showed higher progression rates than in more recent cohorts (Spearman's rho = 0.645, p = 0.034), but this trend did not reach statistical significance in high-risk groups (rho = 0.360, p = 0.272). Meta-analytic data further supported elevated progression in older cohorts. SMM appears to follow a more indolent trajectory in recent years, likely due to improved diagnostic precision and exclusion of biomarker-defined or subclinical MM. Enhanced predictive models may better guide therapeutic decision-making, targeting treatment to those most likely to benefit and avoiding the burden of unnecessary intervention in low-risk individuals. Supported by the Austrian Forum Against Cancer.
Local immunotherapy has the potential to deliver high doses of immune-potentiating agents with limited systemic toxicity. However, clinical development and implementation have been hindered by numerous challenges. Journal for ImmunoTherapy of Cancer will be addressing the potential promise of local immunotherapy and providing guidance on how to overcome current challenges in local drug development through a series of special reports over the next year.
Amyotrophic lateral sclerosis (ALS) is a heritable disorder where rare variants with low-to-moderate penetrance are thought to dominate genetic risk. To identify such rare variants, we harmonized and analyzed exome data from 22 cohorts, totaling 17,919 individuals with ALS and 200,703 controls across discovery and replication phases. Rare variant analyses identified several new risk genes, with replication confirming association of YKT6 and supporting HTR3C, GBGT1 and KNTC1. We also provide strong, independent validation for genes with limited previous evidence: ARPP21, DNAJC7 and CFAP410. Notably, in ARPP21, we identified a new high-effect variant (p.P747L) and confirmed that p.P563L is an ALS-associated variant leading to an aggressive disease course. Beyond new discoveries, our analyses largely recapitulated the known genetic architecture of ALS, identifying risk variants in over 20% of cases and supporting a cumulative oligogenic risk model. These findings highlight new translational targets and show that rare variant analyses capture substantially more genetic risk than common variant genome-wide association studies.
This study is the first to utilize nano-scale Qodume Shirazi seed mucilage (QSSM) and Camelina sativa seed mucilage (CSSM), individually and in combination, as natural wall materials for microencapsulation of Lacticaseibacillus rhamnosus (LR). SEM analysis indicated smooth bead surfaces and successful entrapment of LR. The 50:50 QSSM: CSSM formulation exhibited 97.92% encapsulation efficiency, and microencapsulated LR (MLR) remained viable (>6 log CFU/g) after 15 min at 72 °C. Moreover, MLR maintained viability above 7.85 log CFU/mL for 21 days at 4 °C in goat milk dessert (GMD). Under simulated gastrointestinal conditions MLR demonstrated a 57.53% survival rate compared with free LR, which was 47.17% at the end of storage. The addition of MLR increased the hardness (258.0 ± 16.0 g) and gumminess (102.56 ± 8.45 g) of GMD. Overall, the results demonstrate that QSSM and CSSM mucilages can serve as effective natural encapsulating agents, enhancing the thermal and storage stability.
Monoethylene glycol (MEG) is widely used as a hydrate inhibitor in produced water operations; however, its presence complicates treatment processes by increasing chemical oxygen demand (COD) and posing potential environmental risks to receiving water bodies. This study aimed to isolate and identify efficient MEG-degrading bacteria through systematic screening, followed by whole genome sequencing (WGS) of the top-performing strain to elucidate its biodegradation potential and genetic basis. Among 14 bacterial strains representing 9 genera, Stutzerimonas stutzeri EX72 exhibited the highest degradation performance, achieving a 62.28% reduction in COD within 10 days. Detailed kinetic experiments demonstrated near-complete removal of MEG from the medium within 14 days, confirmed by Gas Chromatography-Flame Ionization Detection (GC-FID). Integrated COD, total organic carbon (TOC), dissolved organic carbon (DOC), and biomass measurements revealed substantial mineralization of MEG, with apparent biomass yields of 0.167-0.188 g biomass per g of MEG-C consumed, indicating efficient carbon partitioning into both biomass formation and respiration. WGS revealed that EX72 encodes pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) responsible for the initial oxidation of MEG to glycolate, followed by assimilation via the glycerate pathway. Phylogenetic and synteny analyses showed that these enzymes cluster with environmental Stutzerimonas/Pseudomonas lineages and possess conserved genomic organization, supporting a conserved catalytic role in MEG oxidation that is mechanistically analogous, though evolutionarily distinct, from well-characterized glycol-degrading systems. Notably, two ADHs (SS.57032 and SS.63794) exhibited 80.58% and 84.91% sequence identity to those encoded by the gene pedH from Pseudomonas putida KT2440 and the ExaA gene from Pseudomonas aeruginosa, respectively. Overall, this study advances understanding of microbial MEG biodegradation in produced water systems and establishes S. stutzeri EX72 as a strong candidate for biological MEG removal, providing a foundation for future investigations into pathway regulation and metabolic fate.
The plans for sustained human presence on the Moon have increased interest in understanding the effects of lunar conditions on terrestrial biology. Plants play a vital role in space exploration, as they not only generate oxygen and remove carbon dioxide but also provide fresh food that can be rich in nutraceutical compounds important for astronaut survival in long-duration missions. Understanding how to leverage in-situ resources, such as lunar regolith, is crucial for sustainable space exploration. However, using regolith as substrate for plant growth is challenging due to its composition and lack of organic matter. While previous research has primarily examined the gene expression of Arabidopsis thaliana under lunar conditions, it has mainly focused on individual gene activity, neglecting the interactions between genes within networks. This study goes beyond traditional approaches by employing differential gene correlation analysis to explore how gene pairs interact across different regolith environments. This strategy provides a deeper understanding of plant adaptation mechanisms, offering valuable insights for optimizing plant growth in extraterrestrial environments.
Type 1 diabetes (T1D) is an autoimmune disease marked by selective destruction of pancreatic β-cells, resulting in absolute insulin deficiency. Although insulin replacement remains the standard therapy, it does not address the underlying autoimmune process or prevent long-term complications. Advances in understanding the pathogenesis have highlighted the interaction of genetic susceptibility, environmental triggers, and immune dysregulation, paving the way for innovative immunotherapies. Current strategies include nonspecific immunosuppressants, monoclonal antibodies (e.g., teplizumab, rituximab), peptide vaccines, and cell-based therapies such as regulatory T cells and stem cells. Among these, teplizumab has gained FDA approval to delay disease onset in high-risk individuals, representing a milestone in preventive intervention. Nevertheless, limited durability, high costs, and safety concerns restrict broader clinical application. Looking forward, personalized treatment strategies, rational drug combinations, and early preclinical interventions are expected to optimize outcomes, offering new hope for improving prognosis and quality of life in T1D patients.
Treatment with brexucabtagene autoleucel (brexu-cel), an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, demonstrated a high objective response rate (93%) and complete response rate (67%) in 60 patients with relapsed/refractory mantle cell lymphoma (R/R MCL) treated in the pivotal ZUMA-2 Cohort 1 study. Subsequently, brexu-cel was approved in the United States and European Union for the treatment of adults with R/R MCL (after ≥ 2 prior therapies in the European Union). Here we report 5-year outcomes from the pivotal ZUMA-2 Cohort 1 study (N = 68), as well as two previously unpublished ZUMA-2 data sets, long-term outcomes in 10 patients who received axi-cel in Cohort 1 and in 14 patients who received a lower dose of brexu-cel in Cohort 2. The primary endpoint for all cohorts of ZUMA-2 was objective response rate. Key secondary endpoints included duration of response (DOR), overall survival (OS), and safety. Patients could transition to a long-term follow-up study after 24 months for monitoring of survival and select adverse events possibly related to brexu-cel. Patients in Cohort 1 received a single infusion of 2 × 106 anti-CD19 CAR T cells/kg (axi-cel or brexu-cel). Patients in Cohort 2 received 0.5 × 106 anti-CD19 CAR T cells/kg (brexu-cel). Median follow-up for the pivotal cohort (N = 68) was 67.8 months (range, 58.2-88.6) with a median DOR of 36.5 months (n = 60), per investigator review. Median OS was 46.5 months (95% CI, 24.5-60.2; N = 68) and was 60.2 months (95% CI, 42.8-not estimable) in patients with complete response (n = 46). The 5-year incidence of cumulative relapse-related and non-relapse-related mortality was 40% (24/60) and 22% (13/60) in responders, respectively. Descriptive outcomes for axi-cel-treated patients (N = 10) and Cohort 2 (N = 14) are reported herein. No Grade 5 cytokine-release syndrome or neurologic events, subsequent T-cell malignancies, or new safety signals were reported in any patient. Patients in ZUMA-2 continued to have durable responses after 5 years of follow-up with predictable long-term safety, supporting the continued use of brexu-cel in R/R MCL. Interpretations of outcomes in axi-cel-treated patients and Cohort 2 are not feasible due to small patient numbers and unmatched baseline characteristics. NCT02601313 and NCT05041309.
Human l-asparaginase 1 (hASNase1) is a promising next-generation candidate for Acute Lymphoblastic Leukemia (ALL) therapy due to its reduced immunogenicity and superior physiological compatibility. However, its recombinant production remains challenging, as expression in Escherichia coli typically results in insoluble aggregates and strong association with host chaperones, limiting biochemical characterization. Here, we established an optimized workflow combining mild induction conditions with tailored solubilization and purification strategies. Incubation with l-asparagine followed by ATP-mediated washing during affinity chromatography significantly improved protein recovery while reducing chaperone co-purification. This approach enabled the isolation of catalytically active hASNase1 displaying allosteric regulation and cooperative substrate binding, consistent with its proposed tetrameric organization. Complementary in silico analyses identified aggregation-prone regions, potentially providing structural insight into the observed expression challenges. Collectively, this study contributes to the development of improved recombinant production strategies for challenging human enzymes in prokaryotic systems.
The high global mortality of hepatocellular carcinoma (HCC) underscores the need for reliable non-invasive diagnostic biomarkers. In this study, transcriptomic analyses were performed on peripheral blood mononuclear cell (PBMC) and tumor datasets from HCC patients to identify differentially expressed genes (DEGs) using an adjusted p-value 〈 0.01 and |log2FC| 〉 1. Functional enrichment analyses revealed predominant immune-related pathways in PBMCs and metabolic pathway dysregulation in tumor tissues. Integration of PBMC and tumor profiles identified STEAP4, EPC1, CLEC1B, and LCN2 as shared DEGs. Survival analyses indicated that elevated expression of STEAP4, EPC1, and CLEC1B was associated with poorer overall survival in HCC patients. Collectively, these findings highlight consistent transcriptional alterations in PBMCs and tumor tissues and suggest that STEAP4, EPC1, and CLEC1B may serve as potential non-invasive biomarkers with diagnostic and prognostic relevance in HCC.
Isobutanol is a promising advanced biofuel alternative to ethanol for advanced biofuels and can be converted into sustainable aviation fuel (SAF) via the alcohol-to-jet (ATJ) process. Recent studies attempted to improve isobutanol production from S. cerevisiae by metabolic engineering; nevertheless, engineered strains, mostly derived from laboratory strains, often encountered difficulties in industrial-scale production. Herein, this present study aims to develop an industrially viable isobutanol-producing strain of S. cerevisiae D3C (isolate G2-3-2, identified in the previous study) with minimal genetic modification. Isolate G2-3-2 was subjected to conventional mutagenesis, resulting in the development of an isobutanol-tolerant strain, IbOH-1. The genomic DNA analysis of IbOH-1 revealed numerous mutations in genes associated with nitrogen starvation response, cell wall biosynthesis and integrity, stress resistance-related amino acid biosynthesis, and the HOG pathway, all of which may collectively enhance tolerance to isobutanol toxicity. The BAT1 gene was afterwards knocked out by CRISPR/Cas9 to improve isobutanol yield. The isobutanol production from IbOH-1bat1∆ strain was investigated in both nutrient-rich YPD and minimal YNB fermentation media, with particular emphasis on optimizing glucose concentration. The highest concentration of isobutanol attained was 3.12 ± 0.068 g/L in 5-L bioreactor (with 3 L working volume), utilizing YNB based medium containing 150 g/L of glucose, supplemented with 8 g/L of peptone, 3 g/L of (NH4)2SO4, 1 g/L of KH2PO4, 0.5 g/L of MgSO4·7H2O, and 0.05 g/L of FeSO4·7H2O. This study provides new insights into improving isobutanol production in S. cerevisiae, highlighting its potential applicability in industrial biofuel processes.
HEK293 is a preferred cellular platform to produce viral vectors including adeno-associated viruses (AAV). However, HEK293 cells were shown to be genomically unstable and many HEK293 cell lines having distinct genotypes and phenotypes have been reported. Here we generated a stable clonal cell line specifically selected for the optimal production of recombinant AAV (rAAV) by the triple plasmid transfection method. Initially over two thousand single cell clones were isolated from a HEK293 polyclonal cell line and evaluated for their growth profile in suspension, doubling time, ability to recover freeze-thaw cycles and transfection efficacy. A selection of clones that met these specific criteria were then screened for their ability to produce high rAAV titers by triple plasmid transfection, yielding one high-performing clone named NBX1P01. This clone was genomically characterized using optical genome mapping and whole genome sequencing and further evaluated for rAAV production capacity across different serotypes and genes of interest (GOI). NBX1P01 was shown to be genomically stable over 55 population doubling levels (PDL), highly transfectable and able to produce rAAV titers similar or higher than those produced by a commercially available HEK293 cell line using the same culture, transfection, harvest and quantification protocol. The ratio of full-to-empty rAAV particles produced by NBX1P01 was two-fold higher than those of the commercial cell line. Long-read sequencing of the encapsidated DNA from the NBX1P01-produced rAAV indicated high levels of genome integrity with minimal levels of contaminants. These results demonstrated the versatility of NBX1P01 cells and their ability to produce high-quality rAAV vectors.
Dengue is a life-threatening mosquito-borne viral disease ranging from mild symptoms to severe hemorrhagic fever. In this study, a recombinant anti-dengue D8 monoclonal antibody was produced in Nicotiana benthamiana and characterized for protein integrity and glycosylation by LC-MS. The plant-derived antibody lacked plant-specific β1,2-xylose and core α1,3-fucose residues, displaying a mammalian-like glycan profile. Functional evaluation showed potent cross-neutralizing activity against all four dengue virus serotypes (DENV1-DENV4), with the strongest activity against DENV4 (FRNT50 < 1 µg/mL), followed by DENV2 (5.82 µg/mL), DENV3 (9.49 µg/mL), and DENV1 (28.68 µg/mL), comparable to the mammalian-produced counterpart. The antibody also bound strongly to NS1 proteins of all serotypes, especially DENV2, and demonstrated higher reactivity than mammalian-derived anti-NS1 antibodies. Collectively, our results provide proof-of-concept that a glycoengineered plant platform can generate a functional D8 antibody with mammalian-like glycosylation, robust NS1 binding, and cross-neutralizing activity against DENV1-4, prompting further evaluation in Fc-dependent assays and in vivo models.
Many diseases have a genetic origin, and analyzing intracellular structures through genetic data yields specific features for the diagnosis and classification of viral disease samples. In this study, 30 types of viruses were analyzed using a graph-based approach on genetic data. Genetic data has been modeled in the form of genomic sequences at the nucleotide scale using the graph theory of complex networks concepts. Degree and eigenvector centrality metrics were employed to extract features. The decision tree was utilized as a machine learning classifier algorithm on the resulting feature space. The results, presented in the form of interpretable rules, enable the classification and identification of virus types from both a binary and multi-class perspective. The model achieved high accuracy and f1 score, which exceeded 99 % on >173,000 samples. Additionally, the feature extraction algorithm demonstrated robust performance across all datasets and classifiers.
Photoautotrophic cyanobacteria convert CO₂ into bioproducts. However, efficient cell harvesting remains a major obstacle. Auto-bioflocculation offers a promising solution. In cultures of Synechocystis sp. PCC 6803, Synechocystis co-flocculated with natural fungal contaminant (identified as Penicillium sp.) into spherical clusters with internal hollow spaces-but only in the presence of erythromycin (EM), and not without EM. Optimized co-cultivation of Synechocystis and Penicillium for five days in BG11 medium (lacking organic compounds) supplemented with 5 µM EM resulted in complete biomass co-flocculation, yielding up to 2.0 g/L. This corresponds to atmospheric CO₂ capture rate of 0.50 g/L/day, representing a 7.9-fold increase in biomass and a 7.0-fold enhancement in CO₂ capture compared to axenic Synechocystis culture. Protein was the major component of the flocculated biomass, accounting for 39-61 % of dry weight. Increasing EM concentrations (from 0.3 to 10 µM) led to an increase in floc diameter from 0.5 to 1.9 mm. Scaling the culture volume from 100 to 200-1000 mL changed the surface texture of the co-flocculates from relatively smooth to rough with thorn-like structures. Transcriptomic analysis indicated that co-flocculation was associated with upregulation of genes involved in S-layer protein synthesis, molecular chaperones, exopolysaccharide polymerization, and pilin regulation. Genes related to minor pilin proteins, pili function, and capsular polysaccharide synthesis were downregulated. Differentially expressed genes between smooth-surfaced and rough-surfaced co-flocs were reported. This bio-flocculation demonstrates low-energy, organic-free strategy that boosts CO₂ capture, highlighting strong potential for carbon-negative bioprocesses. The protein-rich co-flocs offer opportunities for downstream use in animal feed or biorefinery.
Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for adults with relapsed/refractory (R/R) mantle cell lymphoma (MCL) based on the ZUMA-2 cohort 1 (ClinicalTrials.gov identifier: NCT02601313) study in which brexu-cel demonstrated a 93% objective response rate (ORR) and 67% complete response (CR) rate in patients with R/R MCL and previous BTKi therapy (N = 60). Here, we report the primary results of ZUMA-2 cohort 3 (brexu-cel in patients with BTKi-naive R/R MCL). Adults received brexu-cel at 2 × 106 anti-CD19 CAR T cells per kilogram. The primary end point was ORR assessed by independent radiology review committee (IRRC). As of 26 November 2023, 95 patients were enrolled, and 86 received brexu-cel; median follow-up was 15.5 months. The primary end point was met, with a 91% ORR (95% confidence interval [CI], 82.5-95.9; P< .0001; N = 86) and a CR rate of 73% (95% CI, 62.6-82.2). Estimated 12-month progression-free survival (PFS), duration of response, and overall survival (OS) rates were 75%, 80%, and 90%, respectively. Among 95 enrolled patients, the ORR was 82%, the CR rate was 66%, and the 12-month PFS and OS rates (95% CI) were 73% (62.1-80.8) and 85% (75.6-90.7), respectively. Most patients (88%) experienced treatment-related grade ≥3 adverse events, including 4 treatment-related grade 5 events. Consistent with cohort 1, brexu-cel demonstrated a high ORR and similar safety profile. These results support the continued use of brexu-cel in patients with R/R MCL, and consideration in some patients without previous BTKi therapy who have high-risk disease. This trial was registered at clinicaltrials.gov as #NCT04880434.
Neoadjuvant chemotherapy is standard for stage IB-III triple-negative breast cancer (TNBC), with pathological complete response (pCR) strongly associated with survival. Although escalation with platinum and immune checkpoint inhibitors (ICI) improves pCR and long-term outcomes, patients with pCR in control arms of pivotal trials also show favorable outcomes. Whether the regimen leading to pCR impacts long-term survival is largely unknown. We conducted a systematic review and meta-analysis, searching phase II and III trials including early-stage TNBC patients with pCR. A pooled analysis of Kaplan-Meier-derived individual patient data was performed for event-free survival (EFS) and overall survival (OS), with subgroup analyses by treatment regimens. Of 2830 identified publications, 18 trials comprising 3430 patients were included. Neoadjuvant ICI with chemotherapy improved EFS (HR 0.67; 95%CI 0.50-0.89; p < 0.01) compared with chemotherapy-only regimens, with no significant OS difference (HR 0.84; 95%CI 0.50-1.41; P = 0.51). In contrast, EFS and OS were not significantly different regardless of platinum use (HR 0.55; 95%CI 0.20-1.50; P = 0.24 and HR 0.33; 95%CI 0.09-1.22; P = 0.10, respectively). Similarly, anthracycline-containing regimens showed comparable EFS to anthracycline-free regimens (HR 0.86; 95%CI 0.51-1.45; P = 0.58). For patients with pCR after ICI therapy, no benefit of adjuvant ICI for EFS or OS was observed (HR 1.16; 95%CI 0.55-2.44; P = 0.70 and HR 2.91; 95%CI 0.40-21.37; P = 0.29, respectively). These findings suggest that the context in which a pCR is achieved may influence long-term outcomes. Neoadjuvant ICI-based regimens improve EFS in patients with early-stage TNBC and pCR. However, EFS seems not to be impacted by neoadjuvant chemotherapy type.
Antibody-antigen affinity is a key factor in the development of therapeutic antibodies, influencing candidate selection and their clinical efficacy. We used a combination of three label-free biophysical techniques: SPR, ITC, and fluorescence quenching, to rank anti-MICA single-chain variable fragments (scFvs), complemented by ELISA. We evaluated a wild-type (WT) scFv and its Beta mutant across the platforms, complementing this with molecular dynamics simulations and functional assays. The affinity constants (KD) varied depending on the method used, but SPR provided the most precise discrimination. Flow cytometry confirmed stronger binding of the WT scFv to MICA-expressing gastric cell lines, which was consistent with the results obtained using SPR. Simulations indicated greater conformational stability and more favourable antigen contacts for the WT scFv. Together, these results highlight the value of orthogonal measurements for robust affinity assessment and support WT as a promising lead for further development. This benchmark provides practical guidance for selecting affinity assays in discovery pipelines.