Journal of Biotechnology

L-Threonine is an essential amino acid widely used in the feed, food, and pharmaceutical industries, and microbial fermentation is now the dominant route for its production. Further increases in producer titers depend on random mutagenesis coupled with high-throughput screening (HTS), but progress has been limited by the lack of a biosensor with a broad operating range, high sensitivity, and target specificity. Here, we show that L-threonine induces the tryptophan-repressed trp promoter of Escherichia coli, a response that, to our knowledge, has not been described previously. Deletion analysis showed that both the trpO operator and the trpL attenuation element contribute to induction, and that the lowest basal expression occurs only when both elements are present. By optimizing the ribosome-binding site, the promoter -10 region, and TrpR expression, we constructed an L-threonine biosensor on a pACYC backbone. The sensor responded linearly to L-threonine from 0.1 to 30 g/L (R2 = 0.9895), with an induction ratio of up to 51.7-fold. Among the 20 proteinogenic amino acids tested, only L-threonine produced a detectable response. The biosensor distinguished strains with different L-threonine production capacities and guided the directed evolution of homoserine kinase (ThrB) at a conserved motif, yielding variants with brighter reporter fluorescence and increased L-threonine production. This work provides a high-performance biosensor based on native E. coli regulatory elements for screening L-threonine overproducers.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

Background: Long non protein coding (lnc)RNAs are ubiquitous transcripts known to be major regulators of cellular processes in eukaryotes. In diatoms, specific lncRNA expression is tightly correlated to nutrient fluctuations, which suggests function, but their biological relevance remains to be established. In order to fill this knowledge gap, we sought to design a protocol to generate diatom lncRNA knockout mutants by CRISPR-Cas9 enabling subsequent functional studies to shed light into their mechanistic roles. Since lncRNAs do not contain open reading frames that can be targeted by point mutations for the generation of functional knockouts, we designed a multiplexed CRISPR-Cas9 plasmid harboring two independent gRNA for the specific ablation of lncRNA loci in P. tricornutum. As proof of concept, we used our plasmid-based system to target three phosphate depletion responsive lncRNAs. Results: Using the golden gate based uLoop system, we assembled a single plasmid containing all the necessary components to generate CRISPR-Cas9 large genomic ablations of lncRNAs in P. tricornutum using bacterial conjugation. Three intergenic lncRNAs (Linc91, Linc77 and Linc24) responsive to phosphate fluctuations were selected and independently targeted. Around 32% of the exconjugants presented a monoallelic ablation of the targeted loci and 11% presented a biallelic ablation on the first PCR-screening round. Mutated lines, harboring biallelic lncRNAs ablations for each lncRNA targeted, were successfully generated for each of the targeted lncRNAs. Additionally, a first line of phenotypic analysis revealed significant growth rate differences for one of the mutants ({Delta}Lnc24) under both full and phosphate depleted conditions, suggesting its involvement in cell division regulation under nutrient fluctuations. Conclusions: This work provides a simple and efficient method for the generation of lncRNA knock-out mutants in P. tricornutum. Furthermore, this approach can be easily adapted to other microalgae amenable to transformation, enabling the enhancement of our understanding of these important cellular regulatory transcripts in the marine environment.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

Synthetic allopolyploidization is a powerful strategy for generating strains with novel industrially relevant traits. During the design of select, hybridize, evolved and learn cycle, the mitochondrial inheritance is considered neutral and most studies overcome their phenotypic impact. Here, we generated 168 synthetic allotetraploid strains representing all pairwise parental combinations of eight wild Saccharomyces species, and systematically controlled mitochondrial inheritance in these crosses. These allopolyploids were phenotyped across 31 environmental conditions relevant to biotechnology. In ~50% of all condition-strain combinations conditions, allotetraploids exhibited mid-parent heterosis. In approximately 25% of conditions, we found crosses with hybrid vigor, particularly in combinations involving Saccharomyces arboricola x Saccharomyces mikatae . However, allotetraploids were highly unstable and rapidly sporulated. Our analyses revealed that environmental conditions accounted for the largest effects, follow by its interaction with mitotype. However, mitotype alone showed small effects but significantly affected 42% of the tested conditions, underscoring the importance of mitochondrial inheritance as a key determinant of allopolyploid performance. These findings demonstrate that mitochondrial inheritance is a key, environment-dependent determinant of allopolyploid fitness and should be considered alongside nuclear genome composition in the rational design of industrial allopolyploid strains

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Our prior works have demonstrated the therapeutic potential extracellular vesicles (EVs) derived from batch cultures of brain endothelial cells (BECs) owing to their ability to transfer mitochondria to injured recipient cells. However, conventional batch/flask cultures limit scalable and reproducible production of EVs. Here, we evaluated a CELLine AD1000 bioreactor as a platform for sustained production of EVs from mouse b.End3 BECs and compared bioreactor-derived (BR) EVs with EVs obtained conventional batch cultures (BT). Large EVs (lEVs) and small (sEVs) were isolated using a sequential centrifugation protocol and were characterized using dynamic light scattering, nanoparticle tracking analysis, microBCA assay, western blotting, proteomics analysis, and their ability to rescue bioenergetics in oxygen-glucose deprived (OGD) BECs. Bioreactor cultures maintained high cell viabilities (~90%) for up to eight weeks and produced approximately three- to four-fold greater EV protein yields than batch cultures. BR EVs showed reproducible particle characteristics consistent with EV subtypes derived from BT cultures. CD63, CD9 and Arf6 were detected in EV fractions, whereas the mitochondrial outer-membrane protein TOMM20 was enriched in lEVs. Proteomic analyses revealed that bioreactor-derived lEVs retained the mitochondrial cargo signature observed in batch-derived lEVs. Both bioreactor and batch EVs restored relative ATP levels in ODG-exposed BECs. These findings suggest that bioreactor BEC cultures are a scalable approach for generating functionally active BEC-EVs for downstream characterization and therapeutic development.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

The enzymatic oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) is a key step in the sustainable production of polyethylene furanoate (PEF), yet the final oxidation of 5-formyl-2-furancarboxylic acid (FFCA) to FDCA remains the principal bottleneck for industrial application. Although hydroxymethylfurfural oxidases (HMFOs) can catalyze this transformation, naturally occurring enzymes with high FFCA-oxidizing activity remain largely unexplored. Here, we present an iterative computational bioprospecting campaign to identify natural HMFOs with improved activity toward this rate-limiting reaction. Across four successive rounds, the computational workflow evolved from conservative homology-based screening to large-scale sequence mining assisted by machine learning, with each iteration redesigned according to the principal limitations identified through molecular simulations and experimental characterization. This adaptive strategy led to the discovery of several natural HMFOs with substantially improved FFCA oxidation activity, including candidates whose performance under the tested conditions approached that of the engineered benchmark 8BxHMFO. Beyond the identification of promising biocatalysts, the study demonstrates the value of continuous integration of computational prediction and experimental feedback for refining enzyme discovery campaigns, and provides practical insights into balancing catalytic prioritization, sequence diversity, developability, and computational scalability in future simulation-guided bioprospecting efforts.

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

Sinorhizobium meliloti is a nitrogen-fixing soil bacterium that forms a facultative mutualism with its host alfalfa. S. meliloti strains exhibit diverse colony morphologies on solid culture media that span a continuum from dry to mucoid. Studies on how this trait affects symbiosis rely on mutations in exopolysaccharide II production genes; however, whether natural variation in morphology confers a selective advantage on rhizobia in their natural habitats remains unknown. Here, we used 90 mucoid and 27 dry natural isolates to test whether colony morphology is associated with rhizobial fitness in the soil and in the host, or with the nitrogen-fixation benefits they provide to the plant. Colony morphologies were widely distributed across a phylogenetic tree, suggesting that morphological shifts occurred repeatedly and no single genetic variant was strongly associated with this trait. We inoculated soil mesocosms and plants with a mixture of dry and mucoid bacteria. We observed that dry rhizobia have higher fitness, particularly at 22{degrees}C and 32{degrees}C, but not under cold or drought conditions, independent of the initial rhizobial load. Conversely, mucoid bacteria have a modestly higher relative fitness in host nodules, and single inoculations showed that they tend to confer greater benefits to all three alfalfa varieties. Over time, we also observed rare, repeated conversion from mucoid to dry morphologies in soil and host plants. Taken together, our study reveals that morphological variation within a species in a single agricultural field is important for bacterial fitness in the soil and can have cascading effects on the rhizobia's plant benefits.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.

Mesenchymal stromal cells (MSCs) are promising cell therapeutics, but their development as off-the-shelf products requires cryopreservation, which impairs cell function after thaw. Inflammatory licensing enhances MSC therapeutic attributes, yet protocols vary considerably and little is known about how these variables affect MSC metabolism or post-thaw recovery. We characterized mitochondrial and glycolytic flux in umbilical cord (UC) and bone marrow (BM) MSCs licensed with interferon-{gamma} (IFN-{gamma}), tumor necrosis factor- (TNF-), or both. Brief exposure to either cytokine shifted MSC metabolism toward glycolysis and reduced mitochondrial respiration, whereas prolonged exposure led to heterogeneous responses. Conversely, combined IFN-{gamma} and TNF- increased glycolytic flux and respiratory capacity in most donors, with responses detectable after 2 hours and at concentrations below those conventionally used. Cryopreservation impaired aerobic metabolism, followed by donor-dependent recovery. Preconditioning with combined IFN-{gamma} and TNF- partially improved post-thaw metabolism, particularly in BM MSCs, and enhanced early responsiveness to inflammatory challenge, particularly in UC MSCs, as detected by IDO and candidate biomarkers CTSS and C1S. However, donors with improved post-thaw metabolism did not consistently show increased biomarker responses. These findings demonstrate that metabolic fitness and inducible biomarkers capture complementary aspects of post-thaw MSC quality and may inform preconditioning strategies for cryopreserved MSC products.

The unprecedented rise in greenhouse gases such as carbon dioxide (CO2), and their detrimental effects on the atmosphere have intensified the current climate emergency. This challenge has inspired the development of novel carbon capture and utilisation (CCU) technologies, specifically microbial electrosynthesis (MES), for the bioelectrochemical fixation of CO2 into commodity chemical compounds (CCCs) using microbial biocatalysts. In this study, we systematically enriched and maintained robust mixed microbial communities of electroactive bacteria (EAB) from wastewater treatment sludge to serve as biocatalysts for MES. The MES performance of these biocatalysts under different operational conditions in bioelectrochemical systems (BES) reactors were investigated. BES reactors with enriched biocatalysts incubated and maintained under controlled conditions produced higher CCCs yields than those developed at ambient temperatures, while strict anaerobic operations further improved the MES efficiency compared to aerobic conditions. At an applied potential of -1000 mV vs Ag/AgCl, CO2 conversion reached up to 88.11%, significantly outperforming the lower potential (-600 mV) operation. These findings underscore the importance of systematic development of robust and stable biocatalysts, as well as maintaining strict anaerobic conditions, for achieving efficient MES performance, thereby demonstrating the potential of our approach for scalable industrial CCU applications.

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.