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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.
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.
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.
Bauxite residue (BR) is an extreme environment for microorganisms. The aim of the work was to isolate extremophilic microorganisms for further biotechnological applications, such as bioleaching or waste rehabilitation. At the same time, metagenomic analysis was performed to monitor short-term changes in deposited BR. We isolated and identified alkaliphilic and extreme halotolerant strains of Nesterenkonia massiliensis, N. natronophila, Micrococcus luteus, Aspergillus iizukae, Gibellulopsis serrae, and G. nigrescens from Greek and Hungarian BRs. Most strains were siderophore producers, cellulose degraders and produced oxalic and acetic acids. Metagenomic analysis revealed a shift in the most abundant bacterial classes from the freshly produced BR during 1 month and 3 months of storage: from Gammaproteobacteria (29% relative abundance), to Actinomycetes (31%) and Gammaproteobacteria (39%), respectively. Metagenomic analysis showed the presence of Nesterenkonia species. These results highlight the diverse microbiome of BR and underscore its potential as a valuable reservoir of extremophilic microorganisms.
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.
Malaria is primarily caused by Plasmodium parasites and remains a major global health threat due to drug resistance and limited vaccine efficacy. Monoclonal antibodies (mAbs) targeting the conserved NANP repeat region of the circumsporozoite protein (PfCSP), such as MS-1797, represent a promising prophylactic strategy. Here, we demonstrate the plant-based production of MS-1797 in glycoengineered Nicotiana benthamiana ΔXF. Two variants were generated: MS-01 (original MS-1797) and MS-02 (MS-1797-SEKDEL), which yielded up to 457.3 µg/g fresh weight and 415.7 µg/g FW at 6 days post-infiltration. The plant-derived mAbs were obtained in high purity (>90%) and displayed either human core glycans or high mannose structures. Notably, both MS-01 and MS-02 retained antigen specificity in vitro and bound native PfCSP on sporozoites by immunofluorescence assay. These results establish the feasibility of producing functional anti-malarial mAbs in plants and highlight their potential use as affordable reagents for malaria research, diagnostics, and future prophylactic interventions.
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.
Targeted next-generation sequencing (NGS) of cell-free DNA (cfDNA) enables comprehensive molecular profiling and can guide the selection of genotype-targeted therapies. However, the detection of variants derived from clonal hematopoiesis (CH) is a significant confounder in liquid biopsies. Using a training cohort of 426 variants identified in cfDNA NGS from 225 patients with stage I to IV solid tumors, we developed plasma Clonal Hematopoiesis ORigin Detection (plasmaCHORD), a machine learning model that includes fragment-, variant-, and patient-level features to distinguish between tumor and CH origin for each variant detected by liquid biopsies. Model performance was assessed by comparison with the reference origin for each plasma variant determined from matched white blood cell and tumor NGS. Following the locking of the model parameters, we applied plasmaCHORD to an independent validation cohort of 1,418 plasma variants detected in 114 patients with metastatic cancers, as well as to cfDNA NGS from patients enrolled in a prospective clinical trial (NCT05585684). plasmaCHORD predicted tumor origin versus CH origin in the training set with high accuracy (AUC = 0.94). In the independent validation cohort, the locked model maintained similar overall accuracy (AUC = 0.9) and demonstrated significant improvement in accuracy for clinically significant genes. When applied to clinically challenging cases in the context of a precision oncology clinical trial, plasmaCHORD precisely determined variant origin, preventing mismatches with genotype-targeted therapies. plasmaCHORD, a multifeature machine learning model, can significantly enhance the ability to identify bona fide tumor variants in routine plasma-only NGS, addressing a critical need for implementing liquid biopsy-guided therapy by minimizing misinterpretation caused by CH.
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.
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.
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.
Bacillus altitudinis GG-22, isolated from the phyllosphere of agricultural crops, has been identified as a promising biocontrol agent and plant growth-promoting bacterium with substantial potential in sustainable agriculture. In this study, whole-genome sequencing using Illumina technology, combined with ANI analysis, confirmed the strain's classification as B. altitudinis. The genome revealed a rich set of genes involved in biocontrol mechanisms, including the capacity of synthesis of siderophores (schizokinen and bacillibactin-like compounds), the lipopeptide pumilacidin, the bacteriocin pumilarin, alkylpyrones and Bacillus volatiles. In vitro antagonism assays demonstrated significant inhibitory effects against phytopathogenic fungi and oomycetes, such as Verticillium dahliae and Pythium sp., and B. altitudinis GG-22 also showed limited efficacy against bacterial phytopathogens, including Xylella fastidiosa. Transcriptomic profiling of olive trees treated with GG-22 indicated early activation of auxin transport and systemic acquired resistance (SAR) pathways, alongside substantial downregulation of cell wall remodelling genes. These findings suggest that B. altitudinis GG-22 primes plant defence responses and modulates hormonal pathways critical for growth and stress resilience. Future research should prioritize optimizing application strategies and exploring synergies with other microbial agents to fully harness the biocontrol and growth-promoting potential of B. altitudinis GG-22. This strain holds promise for sustainable agricultural practices, particularly in controlling fungal diseases and improving plant performance under stress conditions.
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.
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.
Fermentation has been used for centuries to preserve foods and enhance their sensory, nutritional, medicinal, and commercial qualities, as demonstrated by products such as bread, beer, yogurt, and cheese. Since the 1970s, advances in genetic engineering, together with recent developments in synthetic biology, have transformed fermentation from a largely empirical practice into a highly controlled and precise technological platform. This transition has enabled the targeted biosynthesis of high-value compounds, including specific proteins, enzymes, polysaccharides, and other functional ingredients with broad applications in food and health sectors. This review examines the evolution from traditional fermentation to precision fermentation, highlighting the key technological innovations driving this shift. It critically evaluates the role of precision fermentation in advancing sustainable food production and human health, while addressing its environmental and economic feasibility, compatibility with existing food systems, and practical implementation challenges. The increasing significance of precision fermentation presents important opportunities to enhance global nutrition, support human well-being, and contribute to the development of resilient and sustainable food systems.
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.
Talquetamab, a bispecific antibody targeting GPRC5D, is approved for triple-class exposed (TCE) relapsed/refractory multiple myeloma (RRMM) based on results from MonumenTAL-1. We report updated indirect comparisons of talquetamab vs. real-world physician's choice of treatment (RWPC) in patients with TCE RRMM. External control arms were created for three MonumenTAL-1 cohorts, two without prior T cell redirection (TCR) therapies who received subcutaneously administered talquetamab 0.4 mg/kg weekly (QW; n = 143) or 0.8 mg/kg every other week (Q2W; n = 154) and one with prior B cell maturation antigen (BCMA) TCR (n = 75) who received either schedule (median follow-up [mFU] 38.2, 31.2, and 30.3 months, respectively), from two real-world studies, LocoMMotion (mFU 26.4 months) and MoMMent (mFU 27.1 months). Imbalances in baseline covariates were adjusted using inverse probability weighting and multivariable regression. The relative effectiveness of talquetamab vs. RWPC was estimated for overall response rate (ORR), ≥ very good partial response (VGPR) rate, and ≥ complete response (CR) rate; odds ratios and relative response ratios (RRs) were derived from weighted logistic regression. Hazard ratios (HRs) for duration of response (DOR), progression-free survival (PFS), time to next treatment (TTNT), and overall survival (OS) were estimated using a weighted Cox proportional-hazards model. In the TCR-naïve cohort, talquetamab Q2W had significantly improved outcomes vs. RWPC; RRs were ORR, 2.58; ≥ VGPR, 5.01; ≥ CR, 52.22 and HRs were DOR, 0.52 (p = 0.0011); PFS, 0.47; TTNT, 0.46; OS, 0.35 (all p < 0.0001). Results were similar in the QW cohort. The prior TCR cohort had favorable outcomes with talquetamab vs. RWPC; RRs were ORR, 3.03; ≥ VGPR, 4.88 and HRs were DOR, 0.09, (p = 0.0004); PFS, 0.30 (p < 0.0001); TTNT, 0.26 (p < 0.0001) and OS, 0.37 (p = 0.0020). With longer follow-up, these comparative analyses further demonstrate the clinical benefit of talquetamab over RWPC in patients with TCE RRMM, irrespective of prior TCR therapy. MonumenTAL‑1, ClinicalTrials.gov identifier NCT03399799/NCT04634552; LocoMMotion, ClinicalTrials.gov identifier NCT04035226; MoMMent, ClinicalTrials.gov identifier NCT05160584.
Non-growing cells are commonly encountered in nature and often desired in biotechnological applications to maximize product yields. Such cells exhibit limited protein synthesis, and their metabolic functionality relies on the long-term stability and repair of enzymes and pathways to sustain metabolic activity. However, knowledge of the factors that influence prolonged metabolism is lacking. A biotechnological example is the production of lactic acid. Here we show that prolonged lactic acid formation in non-growing, translationally blocked cells, is not constrained by the intrinsic maximum number of catalytic cycles, but by the metabolic flux. Faster conversion coincided with faster pathway decay and importantly lower cumulative product yield, and vice versa. This behavior is consistent irrespective of whether the flux is altered through manganese addition, changing the cellular ATP demand, or enzyme expression levels. The correlation between flux and pathway decay is relevant for biotechnological applications, and the fitness of growth arrested environmental microorganisms.
Polyneuropathies are common and often require specialist expertise for accurate diagnosis. This study evaluated the diagnostic performance of ChatGPT-4o on real-world polyneuropathy cases, comparing it to peripheral neuropathy specialists and non-specialist neurologists. One hundred cases were selected from two tertiary centers in Milan, Italy. Standardized summaries included clinical, laboratory, and electrophysiological data. ChatGPT-4o was prompted to provide a leading diagnosis, two differentials, and a confirmatory test. Neurologists reviewed the same cases and generated comparable outputs, then could revise their responses after viewing ChatGPT-4o's suggestions. ChatGPT-4o achieved 65.5% leading diagnosis accuracy, comparable to non-specialists (63.0%) but lower than specialists (74.0%, p = 0.002). For differential diagnoses, it outperformed non-specialists (82.0% vs. 77.5%, p = 0.043) and recommended more appropriate tests (68.0% vs. 53.0%, p < 0.001). After reviewing ChatGPT-4o outputs, non-specialists revised their assessments in 21.8% of cases, improving accuracy. ChatGPT-4o shows potential as a diagnostic aid, particularly in non-specialist or resource-limited settings.