Proteinogenic amino acids are important supplements in foods, feeds, and pharmaceuticals. However, the development of cost-efficient and scalable platform downstream processes for multi-ton manufacturing of amino acids is complicated by upstream process heterogeneity resulting in plethora of implementations at laboratory and industrial scale. Here, we review 18 unit operations and analyzed >50 downstream processes reported over six decades to compare separation performance, sustainability and costs, highlighting process advantages and limitations. We find that chromatography is used in >65% of downstream processes, achieving purities >98% in different setups. Sustainable and cost-effective alternatives seem to challenge this position. For example, membrane separations can achieve purities of 60-98% with reduced environmental footprint, costs, and additional options for process integration, mainly for acidic and basic amino acids. Crystallization is frequently used for formulation and purification if >50 g L-1 of amino acids with extreme pI are processed. Options to streamline current processes are discussed.
Bone marrow stromal cells (BMSCs) are classified as Advanced Therapy Medicinal Products (ATMPs) in the subcategory of Cell Therapy Medicinal Products (CTMPs), and the production of these cells for clinical use under good manufacturing practice (GMP) standards is regulated by the European Medicines Agency (EMA). To meet GMP standards for CTMPs, the accuracy and precision of cell-counting methods are essential for monitoring in-process quality and controlling clinical dosing. To achieve this purpose, we validated an automated cell-counting method for evaluating cell concentration and viability, enabling large-scale analysis and reducing analyst-associated variability. Automated cell counting was performed using the LUNA™ automated cell counter, and we validated the parameters against the hemocytometer using the ICH Q2R1 framework. The results showed that automated cell counting yielded acceptable accuracy (90–110%) and RSD (<10%). This demonstrates that LUNA™ automated cell counters may provide an efficient alternative for measuring BMSC cell concentration and viability.
Lactic acid bacteria (LAB), such as Limosilactobacillus fermentum are persistent contaminants in bioethanol fermentation facilities and inhibit Saccharomyces cerevisiae, reducing ethanol yields. Rising resistance to antibiotics such as virginiamycin makes contamination control increasingly difficult. Recombinant endolysins such as LysKB317 provide a promising alternative. To improve LysKB317 performance, five variants were engineered by duplicating and rearranging its enzymatically active (EAD) and cell binding (CBD) domains. These constructs were compared to the wild-type endolysin. Differential scanning calorimetry (DSC) and bacterial lytic assays demonstrated that domain shuffling altered bacteriolytic activity and thermostability. The wild-type LysKB317 exhibited the highest initial lytic activity (1.16 × 10-4 OD/s‧µM), whereas LysKB317EADx3-CBD displayed comparable bacteriolytic kinetics (7.99 × 10-5 OD/s‧µM) and LysKB317CBD-EAD-CBD (ΔCp = 24.66 kJ/mol‧K) exhibited the greatest thermostability. Despite reduced activity, both variants effectively control LAB contaminants and prevented stalled corn mash fermentation. Domain shuffling demonstrated effective bacterial control strategy for generating novel endolysins for biocontrol.
Cadmium (Cd), a toxic heavy metal, poses a significant threat to aquatic ecosystems by impairing primary producers such as microalgae. This study investigated the adaptive responses of Chlamydomonas reinhardtii to Cd stress through physiological, biochemical, and GC-MS-based lipidomic analysis. Cultures were exposed to the EC50 concentration of Cd (20 ppm) for 8 days. Cd exposure significantly increased oxidative stress markers, including hydrogen peroxide (+69%), malondialdehyde (+336%), and proline (+144%). Total carbohydrate and lipid contents increased by 21.7% and 14%, respectively, whereas protein and total photosynthetic pigment contents decreased by 39.5% and 44%, respectively, indicating metabolic reprogramming under Cd stress. Antioxidant capacity was enhanced as reflected by a 50% reduction in the DPPH IC50 value. Lipidomic analysis revealed a decrease in unsaturated fatty acids accompanied by the accumulation of lipid-derived aldehydes and saturated hydrocarbons. KEGG pathway enrichment analysis identified significant alterations in fatty acid biosynthesis, fatty acid elongation, glutathione metabolism, and butanoate metabolism, highlighting coordinated oxidative and lipid metabolic reprogramming as key adaptive responses to Cd stress.
Chinese Hamster Ovary (CHO) cells are used for therapeutic protein production. Although industrial platforms rely on serum-free media, fetal bovine serum (FBS) remains common in research and development. Reducing serum while maintaining cell growth is of interest.In this study, CHO-K1 cells were adapted from 10% to 1% FBS in Ham’s F-12 medium. Six plant protein hydrolysates (wheat, soy, pea, broad bean, rice, and a rice–pea blend) were biochemically characterized and evaluated for their effects on cell proliferation under serum-reduced conditions.CHO-K1 cells tolerated adaptation to 1% FBS, although proliferation decreased compared to 10% FBS. Supplementation with hydrolysates improved cell growth in a concentration-dependent but nonlinear manner, with low concentrations more favorable than higher concentrations. Wheat (W106) and broad bean (F120) peptones showed the strongest effects, partially restoring proliferation toward high-serum conditions.This study provides an evaluation of plant hydrolysates and supports their use as supplements in serum-reduced CHO-K1 culture.
Variable domains of new antigen receptors (VNARs) derived from shark immunoglobulin new antigen receptors (IgNARs) have gained attention as promising single-domain antibody modalities owing to their compact structures, structural stability, and unique binding capabilities. This study aimed to demonstrate the feasibility of screening a synthetic library based on VNARs derived from the brownbanded bamboo shark (Chiloscyllium punctatum) using a complementary DNA (cDNA) display system. A synthetic VNAR library was constructed by randomizing the CDR1 and CDR3 loops, resulting in a genetic diversity of approximately 1012 sequences in the cDNA display format. Using fluorescent proteins as model antigens, in vitro selection was performed, followed by next-generation sequencing and biochemical validation. VNAR-E1 and VNAR-N1 were identified as binding to EGFP and mNeonGreen, respectively. This work represents the first demonstration of applying a cDNA display system to VNAR selection.
Sustainable production of aromatic amino acids (AAAs) and their derivatives are central to developing renewable routes for aroma- and flavour-related chemicals with myriads of applications in pharmaceutical industry, food and nutrition, cosmetics and personal care, polymer synthesis, and biotechnology. The aim of the present study was to establish and validate a high-throughput mass spectrometry (HT-MS) workflow for rapid screening of AAAs and selected aromatic derivatives, i.e., kynurenic acid, indole-3-acetic acid, and p-coumaric acid, using an automated solid-phase extraction system coupled to time-of-flight detection. A method based on fast cycle time, 15 s per sample, and minimal sample handling was elaborated to enable high-throughput screening of large culture collections. Validation using pure standards confirmed excellent linearity (R² ≥ 0.99), accuracy, precision, and on-instrument stability at 4 °C. Although strong matrix effects were observed in microbial culture supernatants, analyte detection remained selective and reproducible, supporting reliable semi-quantitative screening. The results demonstrate that the proposed workflow supports semi-quantitative, comparative analysis of AAAs and their derivatives across large sample sets. Overall, the developed HT-MS method provides a robust and efficient platform for rapid pre-screening of microbial collections accelerating the discovery and optimization of aromatic metabolite production.
Tuberculosis (TB) remains a major global health challenge, emphasizing the need for rapid and reliable diagnostic tools. As technologies move toward simplicity, portability, and real-time analysis, electrochemical biosensors have emerged as promising solutions for effective and sustainable TB detection. This systematic review highlights recent progress in electrochemical immunosensors for Mycobacterium tuberculosis (Mtb). A comprehensive search of PubMed, Scopus, and Web of Science identified 19 relevant studies targeting antigens such as Ag85, ESAT-6, CFP-10, MPT64, LAM, and Hsp16, with one study detecting anti-LAM antibodies. Electrode modifiers were categorized into metallic nanoparticles, conductive polymers, carbon-based nanocomposites, and ternary hybrids. Although carbon-based nanomaterials offer excellent conductivity and biocompatibility, their use in TB immunosensors remains limited. A growing trend toward label-free formats reflects the need for point-of-care compatibility. Advances in nanomaterials have improved sensor performance, but further research using next-generation modifiers and nanozymes is needed to enhance sensitivity and reproducibility.
MAPK and NF-κB pathways regulate macrophage responses to microbial stimuli. Repeated exposure to lipopolysaccharide (LPS) induces endotoxin tolerance, a state in which inflammatory cytokine production is suppressed while antimicrobial functions are preserved. Although regulatory mechanisms of LPS tolerance is well established, how prior LPS exposure reshapes signaling dynamics downstream of TLR4 remains unclear. Using biosensors and live-cell imaging, we quantified ERK and NF-κB signaling in RAW264.7 macrophages during LPS tolerance. Tolerized macrophages produced less TNF-α and IL-6 and showed reduced ERK and NF-κB activity, with lower amplitudes and areas under the curve than cells receiving a single LPS stimulation. Both pathways also displayed delayed activation, reflected by a prolonged time to first peak. Inhibition experiments revealed bidirectional crosstalk, as blocking ERK altered NF-κB signaling and NF-κB inhibition suppressed ERK dynamics. These findings show that LPS tolerance involves coordinated changes in the strength and timing of ERK and NF-κB signaling.
Gynura pseudochina is an Indonesian medicinal herb valued for its flavonoid and anthocyanin content. Despite its pharmacological importance, approaches to enhance metabolite accumulation and clarify associated molecular responses remain limited. This study evaluated 10 nm silver nanoparticles (AgNPs) as elicitors of in vitro growth, metabolite accumulation, gene expression, and sequence-based characterization. We observed dose-dependent modulation of organogenesis and biomass, with improved growth at moderate concentrations (notably 30 µg/L). Flavonoid and anthocyanin contents peaked at 60 µg/L. RAPD profiles remained monomorphic across the tested AgNP concentrations, indicating that no detectable polymorphic variation was observed within the resolution limits of the RAPD markers employed in this study. Partial cDNAs of GpPAL, GpDFR, and GpCHS were successfully isolated, and phylogenetic analysis showed close relationships with Gynura bicolor, with sequence similarities of 92.80%, 100%, and 98.31%, respectively. qRT-PCR revealed strong induction of GpPAL at 60 µg/L, whereas GpDFR and GpCHS were downregulated. Overall, AgNPs effectively modulated growth and phenylpropanoid metabolism, providing a reproducible nano-elicitation framework for medicinal plants with limited genomic resources.
Schizosaccharomyces pombe (S.pombe), commonly referred to as fission yeast, is a widely utilised model organism for studying cellular responses to stress. The stress response mechanisms are largely conserved, offering insights into similar processes in higher eukaryotes, including humans.We have developed an in vivo nanobody-library screening platform in Δmtl2 fission yeast subjected to lethal caspofungin stress, enabling the direct selection of nanobodies that improve survival. Through this method, we isolate a nanobody (Clone-C) that binds to and regulates the Rho1 GEF (Guanine nucleotide Exchange factor) Rgf1, enhancing cell survival and uncovering a hitherto unreported Mtl2–Rgf1–Rho1GTPase regulatory axis in the cell-wall stress response. This nanobody-based fluorescence-readout method offers a versatile tool for gene identification and antifungal target/ADC screening under certain stress conditions.
The recent mpox outbreaks in non-endemic countries highlight the urgent need for improved therapeutics and diagnostics. In this study, monoclonal antibodies (mAbs) targeting the mpox enveloped virion antigens A35 and B6 were transiently expressed in Nicotiana benthamiana using a geminiviral vector system. Following agroinfiltration, anti-A35 and anti-B6 mAbs accumulated to 27 µg/g fresh weight at 3 days and 260 µg/g at 7 days post-infiltration, respectively. SDS-PAGE and Western blot analyses confirmed the assembly of antibodies, and the purified antibodies bound to mpox-infected Vero cells. The neutralization assays demonstrated moderate reductions in viral infection under the tested conditions. Additionally, an electrochemical immunosensor demonstrated the ability of plant-produced antibodies to detect mpox virus through antigen–antibody binding induced current changes. These results support that the plant-based systems as rapid platforms for producing mpox-specific antibodies for diagnostics and antiviral research.
Chronic hepatitis B virus (HBV) infection remains a major global health burden due to the persistence of covalently closed circular DNA (cccDNA), which limits current antiviral therapies. We developed an adeno-associated virus (AAV)-delivered CRISPR/Cas9 system targeting conserved regions of the HBV genome. Three guide RNAs (gRNA1-3) targeting overlapping open reading frames of the surface antigen and polymerase genes were evaluated in HepG2.2.15 cells and HBV-infected hepatocyte-like cells (imHCs), with a reverse transcriptase-targeting gRNA and tenofovir alafenamide as controls. All gRNAs significantly reduced intracellular and extracellular HBV DNA levels and moderately decreased HBsAg secretion. Notably, gRNA2 induced a frameshift mutation and demonstrated superior antiviral efficacy, markedly reducing cccDNA levels, viral DNA levels, viral RNA levels, HBcAg expression, and HBsAg secretion with suppression maintained for up to 12 days. These findings highlight AAV-mediated CRISPR/Cas9 as a promising gene-based therapy for chronic HBV infection.
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.
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.
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.
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.
Polyhydroxybutyrate (PHB) is a biodegradable polymer produced by bacteria under nutrient-limiting conditions, offering a sustainable alternative to petroleum plastics. This study investigated PHB production by bacterial isolates from plastic-contaminated soils. Forty isolates were screened using Sudan Black B, Nile Blue A, and Nile Red; 12 tested positive, and 11 promising strains belonging to genera, Corynebacterium, Bacillus, Micrococcus, Arthrobacter, and Sinomonas were selected for detailed study. Cultivation in nutrient-limiting medium with 2% sugars (fructose, glucose, sucrose, galactose, mannitol) or agro-wastes (cassava wastewater, cassava and potato peels) revealed strain- and substrate-dependent PHB accumulation, peaking at 24–96 h. Micrococcus sp. OO(14)-5 produced the highest PHB content (0.15 g/L) on mannitol, while Corynebacterium sp. FT(1)-6 and Bacillus sp. GO(10)-6 yielded 3.2–3.4 g/L and ∼60% PHB/CDW on cassava wastewater. Other isolates produced >2.7 g/L on peels. Fourier-transform infrared spectroscopy confirmed PHB. These findings highlight low-cost agro-wastes as effective substrates for sustainable PHB production.
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.
