Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Avian pathology is the scientific study of diseases in birds, focusing on the structural, functional and molecular changes in tissues and organs caused by infections, toxins, nutritional deficiencies or others. It plays a critical role in maintaining poultry health, ensuring food security and supporting economic growth. The aim of this review is to highlight current trends, challenges and future directions in avian pathology, with a special emphasis on advancements in diagnostic approaches that enhance avian disease detection and management. However, the practical application of advanced technologies in avian pathology remains limited, particularly in Ethiopia. Recent diagnostic advancements, including immunohistochemistry, molecular techniques as well as digital pathology, have improved the detection, characterisation and management of poultry diseases. Future directions emphasise the use of artificial intelligence (AI) and machine learning for accurate diagnostics, real‐time disease monitoring and outbreak prediction. Ethiopia has achieved significant progress in avian pathology, particularly through polymerase chain reaction and histopathology. Despite ongoing advancements, the poultry industry continues to face challenges, including emerging and re‐emerging pathogens, limited access to diagnostic infrastructure, zoonotic risks and antimicrobial resistance. Therefore, strengthening biosecurity practices, promoting responsible antimicrobial use and expanding the use of molecular, digital pathology and AI‐supported diagnostic tools remain essential strategies for protecting both poultry population and public health. To further enhance disease detection and control, diagnostic capacity and professional training in avian pathology should be strengthened in Ethiopia.

Candidates:
A. The evidence does not state that it plays a critical role in maintaining poultry health, ensuring food security and supporting economic growth.
B. It plays a critical role in maintaining poultry health, ensuring food security and supporting economic growth.
C. The evidence does not state that however, the practical application of advanced technologies in avian pathology remains limited, particularly in Ethiopia.
D. The aim of this review is to highlight current trends, challenges and future directions in avian pathology, with a special emphasis on advancements in diagnostic approaches that enhance avian disease detection and management.
E. Avian pathology is not the scientific study of diseases in birds, focusing on the structural, functional and molecular changes in tissues and organs caused by infections, toxins, nutritional deficiencies or others.
F. The aim of this review is not to highlight current trends, challenges and future directions in avian pathology, with a special emphasis on advancements in diagnostic approaches that enhance avian disease detection and management.
G. However, the practical application of advanced technologies in avian pathology remains limited, particularly in Ethiopia.
H. Avian pathology is the scientific study of diseases in birds, focusing on the structural, functional and molecular changes in tissues and organs caused by infections, toxins, nutritional deficiencies or others.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
This study explores an integrated bioprocessing approach to enhance the bioactivity of Chlorella vulgaris biomass through enzymatic pretreatment and lactic acid fermentation. Three pretreatment strategies—ultrasound-assisted hydrolysis, hydrothermal treatment, and enzymatic hydrolysis using Viscozyme, Alcalase, or their combination- were evaluated for their ability to release fermentable sugars and proteins. Enzymatic hydrolysis proved most effective, yielding up to 151.18 mg g DW − 1 of reducing sugars and 14.4 mg g DW − 1 of protein. Subsequent fermentation with Lactiplantibacillus plantarum and Levilactobacillus brevis demonstrated robust microbial growth (Δ log CFU ≈ 4.0) and significant acidification (pH 3.6–4.2), accompanied by lactic acid production up to 17.35 g/L. Functional characterisation revealed that combined enzymatic pretreatment and fermentation improved antioxidant properties, reaching values of 8.64, 19.46, 1.45 mg Trolox g DW⁻¹ in the CUPRAC, ABTS, and DPPH assays, respectively, while TPC reached 9.67 GAE g DW⁻¹. Moreover, Viscozyme-pretreated, L. plantarum -fermented samples showed inhibitory zones under non-neutralised screening conditions against Gram-positive bacteria. These findings highlight the combined effect of enzymatic pretreatment and microbial fermentation in enhancing the biofunctional value of C. vulgaris , offering a sustainable strategy for developing natural antioxidants and antimicrobial agents for food, nutraceutical, and cosmetic applications. The online version contains supplementary material available at 10.1186/s40643-026-01050-3. Keywords: Microalgae, Chlorella vulgaris , Fermentation, Lactic acid bacteria, Enzyme pretreatment, Antioxidant capacity

Candidates:
A. Subsequent fermentation with Lactiplantibacillus plantarum and Levilactobacillus brevis demonstrated robust microbial growth (Δ log CFU ≈ 4.0) and significant acidification (pH 3.6–4.2), accompanied by lactic acid production up to 17.35 g/L.
B. The evidence does not state that this study explores an integrated bioprocessing approach to enhance the bioactivity of Chlorella vulgaris biomass through enzymatic pretreatment and lactic acid fermentation.
C. Enzymatic hydrolysis proved most effective, yielding up to 151.18 mg g DW − 1 of reducing sugars and 14.4 mg g DW − 1 of protein.
D. Three pretreatment strategies—ultrasound-assisted hydrolysis, hydrothermal treatment, and enzymatic hydrolysis using Viscozyme, Alcalase, or their combination- were evaluated for their ability to release fermentable sugars and proteins.
E. This study explores an integrated bioprocessing approach to enhance the bioactivity of Chlorella vulgaris biomass through enzymatic pretreatment and lactic acid fermentation.
F. Enzymatic hydrolysis proved most effective, yielding up to 152.18 mg g DW − 1 of reducing sugars and 14.4 mg g DW − 1 of protein.
G. Three pretreatment strategies—ultrasound-assisted hydrolysis, hydrothermal treatment, and enzymatic hydrolysis using Viscozyme, Alcalase, or their combination- were not evaluated for their ability to release fermentable sugars and proteins.
H. Subsequent fermentation with Lactiplantibacillus plantarum and Levilactobacillus brevis demonstrated robust microbial growth (Δ log CFU ≈ 5.0) and significant acidification (pH 3.6–4.2), accompanied by lactic acid production up to 17.35 g/L.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Cultivated meat represents a substitute for traditional livestock farming through the external cultivation of animal cells. The technology is still in its infancy and requires continued research and development to achieve commercial viability. This analysis offers an overview of cultivated meat's current standing by examining its nutritional value and safety and comparing it with traditional meat options. The study examines both commercial viability and regulatory hurdles for market entry as well as consumer acceptance and psychological obstacles to adoption. The discussion encompasses food safety concerns, production costs, market opportunities, global regulatory approaches, and industry‐leading company trends in the cultivated meat field. The analysis presents key technological challenges and solutions while examining changes in consumer mindsets, besides sustainability and ethical issues, which remain crucial yet evolving aspects of cultivated meat development. The expanding global population has led to cultivated meat being recognized as a vital sustainable solution for future food security.

Candidates:
A. The evidence does not state that cultivated meat represents a substitute for traditional livestock farming through the external cultivation of animal cells.
B. This analysis offers an overview of cultivated meat's current standing by examining its nutritional value and safety and comparing it with traditional meat options.
C. The evidence does not state that this analysis offers an overview of cultivated meat's current standing by examining its nutritional value and safety and comparing it with traditional meat options.
D. The evidence does not state that the study examines both commercial viability and regulatory hurdles for market entry as well as consumer acceptance and psychological obstacles to adoption.
E. The study examines both commercial viability and regulatory hurdles for market entry as well as consumer acceptance and psychological obstacles to adoption.
F. The technology is not still in its infancy and requires continued research and development to achieve commercial viability.
G. The technology is still in its infancy and requires continued research and development to achieve commercial viability.
H. Cultivated meat represents a substitute for traditional livestock farming through the external cultivation of animal cells.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
This article showed the data of ocular clinical parameters, tear fluid cytokines, and topical cyclosporine-A 0.1% (Ikervis®) compliance in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients. Study visits were conducted at 3–5 weeks pre-allo-HSCT (screening), pre-allo-HSCT, and 3/6/12 months post-allo-HSCT, with data collected at each time point. At each visit, tear samples were assessed for 96 cytokines by the Proximity Extension Assay O-linked target 96 platform.

Candidates:
A. Study visits were conducted at 3–5 weeks pre-allo-HSCT (screening), pre-allo-HSCT, and 3/6/12 months post-allo-HSCT, with data collected at each time point.
B. At each visit, tear samples were assessed for 97 cytokines by the Proximity Extension Assay O-linked target 96 platform.
C. This article showed the data of ocular clinical parameters, tear fluid cytokines, and topical cyclosporine-A 0.1% (Ikervis®) compliance in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients.
D. At each visit, tear samples were assessed for 96 cytokines by the Proximity Extension Assay O-linked target 96 platform.
E. This article showed the data of ocular clinical parameters, tear fluid cytokines, and topical cyclosporine-A 1.1% (Ikervis®) compliance in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients.
F. Study visits were conducted at 4–5 weeks pre-allo-HSCT (screening), pre-allo-HSCT, and 3/6/12 months post-allo-HSCT, with data collected at each time point.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance. A lot‐to‐lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge. This study examined the use of bind‐and‐elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub‐pools from a mixed‐mode chromatography resin‐packed column. Filterability of each sub‐pool through a virus‐retaining filter was subsequently examined. Circular dichroism and Fourier transform infrared spectroscopy were performed for each sub‐pool to probe for higher‐order structure differences between mAb variants enriched therein. Bioanalytical techniques were also used to assess colloidal stability, surface hydrophobicity, surface charge, and size differences. Results showed that basic charge variants, high‐mannose glycovariants, high relative hydrophobicity proteoforms, and high‐molecular‐weight species were enriched in the last‐eluting (terminal) sub‐pools. The first sub‐pool and the final sub‐pool showed the most fouling propensity on VPro virus filters. Circular dichroism showed that enriched proteoforms in the last sub‐pool possessed a higher percentage of bends. Most secondary structures did not vary significantly between sub‐pools. Diffusion interaction parameter was highly negative across all sub‐pools and the bulk unfractionated pool. These results provide a design space for identifying and depleting problematic mAb variants before the crucial virus filtration step. Keywords: chromatography, fractionation, mAbs, proteoforms, relative hydrophobicity variants

Candidates:
A. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge.
B. The evidence does not state that this study examined the use of bind‐and‐elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub‐pools from a mixed‐mode chromatography resin‐packed column.
C. A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance.
D. A rapid assessment of manufacturability for drug candidates is not crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance.
E. A lot‐to‐lot approach to manufacturability is not adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool.
F. A lot‐to‐lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool.
G. This study examined the use of bind‐and‐elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub‐pools from a mixed‐mode chromatography resin‐packed column.
H. The evidence does not state that manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Cas‐CLOVER is an emerging high‐fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas‐CLOVER to establish a robust, gene‐edited platform in suspension‐adapted CHO‐K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high‐yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas‐CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas‐CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas‐CLOVER's dual‐guide RNA design, we generated a GS5 single knockout (GS5‐SKO) and subsequently a double knockout (GS‐DKO) line at both the GS5 and GS1 loci, both with none detected off‐target mutations analyzed in 40 predictably off‐target sites. For functional validation, these cell lines were engineered with the proprietary Harbor‐IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS‐DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high‐producing clones with cell‐specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well‐plate fed‐batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas‐CLOVER as a versatile genome editing tool for developing high‐yield CHO host platforms in CLD. Keywords: bioprocessing, Cas‐CLOVER, cell line development, CHO cells, gene editing, Harbor‐IN transposase, trastuzumab

Candidates:
A. In this study, we applied Cas‐CLOVER to establish a robust, gene‐edited platform in suspension‐adapted CHO‐K2 cells supporting cell line development (CLD) for biopharmaceutical production.
B. An attractive strategy for high‐yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells.
C. Cas‐CLOVER is not an emerging high‐fidelity genome editing system that enables precise and efficient cell engineering.
D. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1).
E. In this study, we applied Cas‐CLOVER to establish a robust, gene‐edited platform in suspension‐adapted CHO‐K1 cells supporting cell line development (CLD) for biopharmaceutical production.
F. Cas‐CLOVER is an emerging high‐fidelity genome editing system that enables precise and efficient cell engineering.
G. An attractive strategy for high‐yield clone selection is not the use of glutamine synthetase (GS) knockout CHO cells.
H. The primary GS gene resides on chromosome 6 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1).


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["100%", "20 nm", "51 nm", "99%", "186 L", "50 nm", "185 L", "21 nm"]

Evidence:
As adeno‐associated viral vectors (AAV) continue to advance through the clinical pipeline, effective downstream purification strategies must be developed to ensure bulk drug purity and safety. AAV are produced within mammalian cells, bringing forth risks associated with viral contamination. Although existing downstream operations provide some degree of viral inactivation and removal, regulatory agencies have recommended the incorporation of a dedicated virus removal filtration step to ensure robust viral clearance. Recently published studies have demonstrated that membrane filters with nominal pore sizes between 35 and 50 nm can provide effective AAV transmission while removing larger viruses, although these results were obtained over a limited range of conditions. This study represents the first investigation into the effects of filtrate flux and process disruptions on virus reduction filtration for AAV. Experiments were performed using purified AAV capsids and carboxylate‐modified polymeric nanoparticles with a nominal diameter of 20 nm. Initial results confirmed that both systems exhibited nearly identical transient transmission profiles during virus filtration. Virus filtration performed at various filtrate fluxes (between 20 and 185 L/m 2 /h) revealed that moderately higher AAV yield may be obtained at lower fluxes. The data were analyzed using a modified internal polarization model, which was extended to account for the effects of process disruptions on transient particle transmission and recovery. Process disruptions were employed to increase AAV yield beyond 99% without compromising overall clearance of large viruses. At least a 4‐log reduction in xenotropic murine leukemia virus (XMuLV) was observed under all conditions tested, even following multiple process pauses. Keywords: adeno‐associated virus (AAV), downstream processing, internal polarization, process disruptions, viral clearance, virus filtration


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
CAR T-cell therapy has become a transformative modality in oncology, demonstrating sustained clinical efficacy in hematologic malignancies. Recent investigations have expanded its potential beyond cancer to immune-mediated disorders, including autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus, as well as chronic viral infections including HIV and hepatitis B. This review examines the mechanistic foundations of CAR-T cells, advances in universal and allogeneic engineering strategies designed to mitigate graft-versus-host disease and host rejection, and emerging in vivo gene-delivery platforms that aim to bypass conventional ex vivo manufacturing. We further evaluate safety-control architectures, including logic-gated and inducible systems, and discuss translational barriers related to scalability, manufacturing standardization, and long-term immune durability. While technological innovations in genome editing, synthetic biology, and computational design continue to refine CAR-T platforms, substantial biological and logistical challenges remain. A critical synthesis of these evolving strategies is necessary to distinguish incremental optimization from paradigm-shifting advances and to define the future trajectory of CAR-based immunotherapy across oncology and immune-mediated diseases.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B186 and muSEAP , separated from PD-L1/2 by a self-cleaving P2A peptide. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L1/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice. Vectors (rAAV6-muSEAP, rAAV6-muSEAP-PD-L1, or rAAV6-muSEAP-PD-L2) were injected into two sites of the gastrocnemius muscle at a total dose of 1×10 10 vg. Co-delivery of PD-L1, particularly, significantly enhanced muSEAP secretion into the bloodstream up to 12 weeks despite elevated anti-AAV6 responses in pre-immunized mice. muSEAP secretion increased 33.3- and 31.4-fold with the co-delivery of PD-L1, while the increase was only 5.6- and 9.3-fold in the muSEAP control group at 5 and 12 weeks, respectively. Ligand-treated pre-immunized animals also had less T-cell infiltration into the treated muscle compared to naïve animals. In summary, co-delivery of PD-L1/2 alongside a transgene represents a promising strategy for achieving sustained gene expression in individuals pre-exposed to AAV.

Candidates:
A. Immune responses against recombinant adeno-associated virus (rAAV) are not one of the major obstacles in gene therapy.
B. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L2/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice.
C. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B187 and muSEAP , separated from PD-L1/2 by a self-cleaving P2A peptide.
D. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance.
E. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L1/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice.
F. We investigated the potential of Programmed Death 2 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance.
G. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B186 and muSEAP , separated from PD-L1/2 by a self-cleaving P2A peptide.
H. Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
In this study we investigated the impact of a phage cocktail on Daphnia magna microbiome and the life‐history parameters. A mixture of four phages able to infect strains of Klebsiella pneumoniae , Enterobacter sp. and Pseudomonas aeruginosa was tested on three D. magna clones . The host‐associated microbiome composition in both the examined variants and the control was analysed using 16S rRNA amplicon sequencing. Additionally, the survival, growth rate, age, size at the first reproduction, and neonate per female were assessed. The analysis revealed minor shifts in microbial composition following phage exposure. Nevertheless, results showed that the phage cocktail increased microbiome diversity. None of the life‐history parameters studied were affected by the presence of the phage cocktail, and no adverse effects were observed. The results indicated that under laboratory conditions the phage cocktail is safe for D. magna and its microbiome.

Candidates:
A. The evidence does not state that a mixture of four phages able to infect strains of Klebsiella pneumoniae , Enterobacter sp.
B. The host‐associated microbiome composition in both the examined variants and the control was analysed using 17S rRNA amplicon sequencing.
C. and Pseudomonas aeruginosa was tested on three D.
D. The evidence does not state that in this study we investigated the impact of a phage cocktail on Daphnia magna microbiome and the life‐history parameters.
E. The host‐associated microbiome composition in both the examined variants and the control was analysed using 16S rRNA amplicon sequencing.
F. In this study we investigated the impact of a phage cocktail on Daphnia magna microbiome and the life‐history parameters.
G. A mixture of four phages able to infect strains of Klebsiella pneumoniae , Enterobacter sp.
H. and Pseudomonas aeruginosa was not tested on three D.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Saponins are a structurally diverse plant glycosides with important ecological functions and broad pharmaceutical and industrial value. Recent advances have shifted saponin research from descriptive pathway elucidation toward predictive and programmable biomanufacturing. High-quality genome assemblies, integrated multi-omics profiling, and metabolic gene cluster analyses have clarified the enzymatic logic and regulatory architecture underlying saponin biosynthesis and structural diversification, enabling quantitative modeling of pathway flux and identification of key regulatory bottlenecks. Building on these foundations, synthetic biology tools, including CRISPR-based transcriptional modulation, synthetic promoters, and transcription factor rewiring, allow precise and programmable control of biosynthetic networks. In parallel, structure-guided enzyme engineering and AI-assisted protein design accelerate the optimization of cytochrome P450s and glycosyltransferases, improving catalytic efficiency and pathway robustness. These strategies are implemented across multiple production platforms, including engineered microbes, plant suspension cells, hairy roots, and adventitious root systems, enabling iterative optimization through Design-Build-Tes-Learn-cycles. Together, this review synthesizes recent conceptual and technological advances, positioning saponins as a model system that bridges gene networks, regulatory logic, and industrial biomanufacturing, and highlighting a generalizable framework for predictive design and scalable production of complex plant natural products.

Candidates:
A. Saponins are a structurally diverse plant glycosides with important ecological functions and broad pharmaceutical and industrial value.
B. The evidence does not state that building on these foundations, synthetic biology tools, including CRISPR-based transcriptional modulation, synthetic promoters, and transcription factor rewiring, allow precise and programmable control of biosynthetic networks.
C. Recent advances have shifted saponin research from descriptive pathway elucidation toward predictive and programmable biomanufacturing.
D. The evidence does not state that high-quality genome assemblies, integrated multi-omics profiling, and metabolic gene cluster analyses have clarified the enzymatic logic and regulatory architecture underlying saponin biosynthesis and structural diversification, enabling quantitative modeling of pathway flux and identification of key regulatory bottlenecks.
E. Saponins are not a structurally diverse plant glycosides with important ecological functions and broad pharmaceutical and industrial value.
F. High-quality genome assemblies, integrated multi-omics profiling, and metabolic gene cluster analyses have clarified the enzymatic logic and regulatory architecture underlying saponin biosynthesis and structural diversification, enabling quantitative modeling of pathway flux and identification of key regulatory bottlenecks.
G. Building on these foundations, synthetic biology tools, including CRISPR-based transcriptional modulation, synthetic promoters, and transcription factor rewiring, allow precise and programmable control of biosynthetic networks.
H. The evidence does not state that recent advances have shifted saponin research from descriptive pathway elucidation toward predictive and programmable biomanufacturing.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Microalgae are increasingly recognized as industrial biotechnology platforms for the sustainable production of high-value products (HVPs), including carotenoids, polyunsaturated fatty acids (PUFAs), polysaccharides, and phycobiliproteins. Their transition from exploratory biomass resources to precision metabolite-focused biofactories reflects growing industrial and regulatory consolidation. This review adopts a product-oriented perspective that moves beyond biomass productivity to evaluate how biological regulation, cultivation strategies, downstream processing, safety assessment, and regulatory readiness collectively determine industrial feasibility and commercialization potential. Rather than treating these components independently, the analysis frames them as interconnected determinants within a systems-level bioindustrial design framework. Emphasis is placed on stress-responsive biosynthesis, extraction and purification bottlenecks, mixotrophic cultivation, and strain engineering approaches as key enablers for improving productivity, process robustness, and cost performance. Carotenoids and omega-3 PUFAs are identified as the most industrially mature microalgal HVPs, supported by scalable production systems and regulatory acceptance, whereas polysaccharides and phycobiliproteins are highlighted as emerging products with expanding bioindustrial relevance. This comparative positioning underscores that industrial maturity depends not solely on biological potential, but on coordinated optimization across strain design, cultivation stability, downstream compatibility, and compliance pathways. By integrating technological innovation with safety and regulatory considerations, this review provides a coherent framework to support scalable, compliant, and sustainable microalgal biomanufacturing. Overall, the synthesis advances a rational roadmap for translating microalgal HVPs from laboratory optimization to economically viable and regulation-ready industrial deployment.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["72 %", "26 %", "233 h", "73 %", "27 %", "22 %", "21 %", "234 h"]

Evidence:
Bio-based carboxylic acids are key platform chemicals for a circular economy, offering sustainable alternatives to fossil-derived products. Yet, high substrate and processing costs, along with narrow profit margins, restrict industrial-scale bioproduction. In situ product removal (ISPR) holds the potential to increase productivity and yield in fermentations by circumventing product inhibition. Thus, it presents a promising process intensification measure to bridge the commercial gap between bio-based and petrochemical platform chemicals. One effective method for product recovery is reactive extraction with trioctylamine. In the present study, this method was applied to itaconic acid (ITA) fermentations with Ustilago cynodontis . First, the successful operation of dispersion-based apparatuses for reactive extraction was proven using small-scale mixer–settlers, with 1-octanol and the biocompatible 2-octanone as diluents. We then developed an improved feeding profile, lowering byproduct formation by 72 %. Subsequently, we demonstrated the feasibility of ISPR using a perfusion bioreactor with an external membrane coupled to reactive extraction and back-extraction. After 233 h of fermentation, the total amount of ITA produced was increased by 26 %, and productivity was 21 % higher compared to extended-batch fermentations. However, the yield was only slightly improved by 5 %. Ultimately, we identified product toxicity far below the maximum titer of 80 g \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hbox {L}^{-1}$$\end{document} L - 1 as a key bottleneck in ISPR fermentations with U. cynodontis . The results underscore the potential of ISPR and warrant further investigation in this field.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Microbial proteases represent critical biocatalysts for industrial applications, yet optimizing their production remains challenging. This study isolated and characterized a high-yield protease-producing bacterium from environmental sources and systematically optimized production parameters. Among 124 bacterial isolates screened from six environmental sources, poultry waste exhibited the highest microbial diversity, yielding 63 morphologically diverse isolates. The most potent proteolytic isolate, P15, was identified through 16 S rRNA gene sequencing as Bacillus velezensis AZH3S (GenBank accession PX417380 ), sharing 99% sequence similarity with B. amyloliquefaciens . Initial screening of eight culture media identified glucose-casein-yeast extract medium as optimal, producing 442 U/mL protease activity. Time-course analysis revealed maximum enzyme production at 24 h (420 U/mL), with yield coefficients increasing to 480 U/g biomass in late stationary phase. Carbon and nitrogen source optimization demonstrated that starch (470 U/mL) and peptone (480 U/mL) were superior substrates. Response surface methodology employing Box-Behnken design across five variables (starch, peptone, pH, temperature, agitation speed) generated a highly predictive model (R² = 0.99) identifying agitation speed as the most influential parameter. Optimized conditions (9.84 g/L starch, 5.46 g/L peptone, pH 7.5, 33.1 °C, 244 rpm) achieved 1160.3 ± 9.20U/mL protease activity, representing a 2.80-fold improvement over initial production. Three-step purification achieved 5.82-fold enrichment with 32.0% yield. The purified enzyme exhibited a molecular mass of approximately 20 kDa with exceptional substrate affinity (Km = 0.207 mM, Vmax = 39.8 µmol/min), following classical Michaelis-Menten kinetics.These findings establish a robust bioprocess for industrial-scale protease production with significant biotechnological potential. The online version contains supplementary material available at 10.1186/s12866-026-04782-6.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Space-based biomanufacturing has historically focused on long-duration crewed and exploration missions, but its greater potential lies in supporting in-space logistics, manufacturing, and servicing in Earth orbit. This article provides a strategic perspective on how shifting investments, policy, and R&D to orbital biomanufacturing could revolutionize defense, commercial, and civil sectors by enhancing supply chain resiliency, operational flexibility, ethical debris management, and commercial viability in Earth orbit.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Background: Bacterial cellulose (BC), natively synthesized by Komagataeibacter spp., is a biodegradable biomaterial with superior mechanical properties. However, under agitated cultivation, cellulose-producing strains (Cel + ) often transition to nonproducing mutants (Cel − ), restricting scalability and hindering widespread use. Agitation-associated shear stress, elevated oxygen levels, and genetic mutations have been linked to the emergence of the Cel − phenotype. A genome-wide investigation that considers population heterogeneity and dynamics is essential to reveal the mutational landscape and evolutionary processes driving this phenotypic shift. Results: Over successive rounds of agitated cultivation, K. intermedius ENS15 transitioned to a planktonic state, losing BC production. Whole-genome sequencing revealed both structural variations (SVs) and nonstructural variations (NSVs). Contrary to the previous reports, SVs, including insertion sequence-mediated junction events, did not affect the genes related to BC synthesis. Instead, the accumulation and positive selection of NSVs, such as frameshift and replication slippage events in key BC-related genes, strongly correlated with the loss of cellulose synthesis. Conclusions: This study provides the first genome-wide population-level analysis revealing mutational dynamics underlying the BC phenotypic switch in Komagataeibacter spp. cultivated under agitated conditions. We show that the BC-related gene mutations are not solely driven by SVs, with NSVs emerging as equally critical contributors. Furthermore, genomic evidence suggests possible involvement of regulatory mechanisms, such as quorum sensing and cyclic dimeric guanosine monophosphate signaling, prompting future studies on regulatory processes under agitated cultivation conditions. These findings highlight the importance of examining genetic heterogeneity to understand phenotypic adaptation for strain improvement strategies.

Candidates:
A. A genome-wide investigation that considers population heterogeneity and dynamics is essential to reveal the mutational landscape and evolutionary processes driving this phenotypic shift.
B. Agitation-associated shear stress, elevated oxygen levels, and genetic mutations have been linked to the emergence of the Cel − phenotype.
C. Background: Bacterial cellulose (BC), natively synthesized by Komagataeibacter spp., is a biodegradable biomaterial with superior mechanical properties.
D. The evidence does not state that agitation-associated shear stress, elevated oxygen levels, and genetic mutations have been linked to the emergence of the Cel − phenotype.
E. The evidence does not state that however, under agitated cultivation, cellulose-producing strains (Cel + ) often transition to nonproducing mutants (Cel − ), restricting scalability and hindering widespread use.
F. Background: Bacterial cellulose (BC), natively synthesized by Komagataeibacter spp., is not a biodegradable biomaterial with superior mechanical properties.
G. A genome-wide investigation that considers population heterogeneity and dynamics is not essential to reveal the mutational landscape and evolutionary processes driving this phenotypic shift.
H. However, under agitated cultivation, cellulose-producing strains (Cel + ) often transition to nonproducing mutants (Cel − ), restricting scalability and hindering widespread use.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Cells integrate exogenous and endogenous signals to grow, repair, or die. This is likely achieved through dynamic functional associations between genes, but measuring these relationships at scale is non-trivial. Here, we evaluate genetic associations in response to cell-cycle interruption, genotoxic perturbation, and nutrient deprivation using conditional genetic interaction (GI) mapping in human cells. In five maps measuring 250,000 GIs or higher-order environmental interactions, we discover widespread rewiring of relationships between genes, complexes, and ontologies across conditions. Specific bioprocesses drive the rewiring signal in each environmental state, as highlighted in our findings that the TIP60 and PP2A complexes radically alter their interaction profiles after inhibition of ATR. This resource reveals numerous genetic relationships for the fields of DNA damage signaling, DNA repair, and cell-cycle control and explores their context specificity. Our work advances a framework for using GI maps to explore environmental rewiring.

Candidates:
A. Cells integrate exogenous and endogenous signals to grow, repair, or die.
B. This is likely achieved through dynamic functional associations between genes, but measuring these relationships at scale is non-trivial.
C. This is not likely achieved through dynamic functional associations between genes, but measuring these relationships at scale is non-trivial.
D. In five maps measuring 251,000 GIs or higher-order environmental interactions, we discover widespread rewiring of relationships between genes, complexes, and ontologies across conditions.
E. Here, we evaluate genetic associations in response to cell-cycle interruption, genotoxic perturbation, and nutrient deprivation using conditional genetic interaction (GI) mapping in human cells.
F. The evidence does not state that here, we evaluate genetic associations in response to cell-cycle interruption, genotoxic perturbation, and nutrient deprivation using conditional genetic interaction (GI) mapping in human cells.
G. In five maps measuring 250,000 GIs or higher-order environmental interactions, we discover widespread rewiring of relationships between genes, complexes, and ontologies across conditions.
H. The evidence does not state that cells integrate exogenous and endogenous signals to grow, repair, or die.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
This data article presents a labelled flow-based network traffic dataset collected from a controlled Internet of Things (IoT) laboratory environment. The dataset captures network communication generated by Raspberry Pi-based IoT nodes configured to emulate service and client roles. Traffic was recorded during normal operations and during the execution of predefined cyberattack scenarios within an isolated experimental network. Network traffic was recorded at the packet level using passive network monitoring and stored in PCAPNG format. The packet captures were subsequently processed into bidirectional network flows, producing flow records with statistical and temporal attributes derived from the observed packet exchanges. Cyberattack-related flows were labelled using the experimental ground-truth markers recorded during each attack campaign, complemented by the fixed attacker node IP address. Flows outside the marked intervals were labelled as benign and corresponded to regular device communication. This combined labelling approach reduces the potential for overlap between benign and attack activities. The dataset covers nine attack scenarios grouped into six attack categories. It is released through a structured repository containing raw packet captures, labelled flow files, and supporting metadata for flow-based IoT traffic analysis, cyberattack detection research, and optional re-labelling.

Candidates:
A. The evidence does not state that this data article presents a labelled flow-based network traffic dataset collected from a controlled Internet of Things (IoT) laboratory environment.
B. This data article presents a labelled flow-based network traffic dataset collected from a controlled Internet of Things (IoT) laboratory environment.
C. The evidence does not state that the dataset captures network communication generated by Raspberry Pi-based IoT nodes configured to emulate service and client roles.
D. Network traffic was recorded at the packet level using passive network monitoring and stored in PCAPNG format.
E. Traffic was not recorded during normal operations and during the execution of predefined cyberattack scenarios within an isolated experimental network.
F. Network traffic was not recorded at the packet level using passive network monitoring and stored in PCAPNG format.
G. The dataset captures network communication generated by Raspberry Pi-based IoT nodes configured to emulate service and client roles.
H. Traffic was recorded during normal operations and during the execution of predefined cyberattack scenarios within an isolated experimental network.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Hypocrellin A (HA), a photoactive perylenequinone from the bambusicolous Shiraia fungi , possesses potent photodynamic anticancer and antimicrobial properties. However, the signaling mechanisms governing its biosynthesis remain poorly understood. In this study, we identify spermidine (Spd), a ubiquitous polyamine, as a novel elicitor that significantly enhances HA production in Shiraia sp. S9. Spd activated both nitric oxide synthase (NOS) and nitrate reductase (NR) for nitric oxide (NO) generation, leading to the stimulation of the soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signaling cascade. Inhibition of NO generation or sGC activity suppressed both cGMP accumulation and HA biosynthesis. Transcriptomic analysis revealed that Spd-induced NO signaling upregulated genes in central carbon metabolism and the hypocrellin biosynthetic gene cluster. The dual elicitation strategy by the combined addition of Spd and the NO donor sodium nitroprusside (SNP) exhibited a strong enhancing effect, increasing HA yield by 4.6-fold compared with control cultures. These results demonstrate that Spd regulates HA biosynthesis through a NO–cGMP–mediated signaling pathway, unveiling polyamines as new metabolic elicitors and providing an efficient dual-elicitation strategy for large-scale hypocrellin production. The online version contains supplementary material available at 10.1186/s40643-026-01051-2.

Candidates:
A. The evidence does not state that hypocrellin A (HA), a photoactive perylenequinone from the bambusicolous Shiraia fungi , possesses potent photodynamic anticancer and antimicrobial properties.
B. Spd activated both nitric oxide synthase (NOS) and nitrate reductase (NR) for nitric oxide (NO) generation, leading to the stimulation of the soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signaling cascade.
C. However, the signaling mechanisms governing its biosynthesis remain poorly understood.
D. In this study, we identify spermidine (Spd), a ubiquitous polyamine, as a novel elicitor that significantly enhances HA production in Shiraia sp.
E. The evidence does not state that spd activated both nitric oxide synthase (NOS) and nitrate reductase (NR) for nitric oxide (NO) generation, leading to the stimulation of the soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signaling cascade.
F. Hypocrellin A (HA), a photoactive perylenequinone from the bambusicolous Shiraia fungi , possesses potent photodynamic anticancer and antimicrobial properties.
G. The evidence does not state that in this study, we identify spermidine (Spd), a ubiquitous polyamine, as a novel elicitor that significantly enhances HA production in Shiraia sp.
H. The evidence does not state that however, the signaling mechanisms governing its biosynthesis remain poorly understood.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Bioprocesses grant us with broad opportunities to build a circular carbon economy. Using microbes allows us to replace non-renewable resources and change waste management strategies, including recycling and upcycling. However, research still lags on strategies to enable economic viability of these types of bioprocesses relative to the traditional production methods. Tuning the composition of the microbial growth medium is one way to address these issues. This allows us to gain insight into the interactions of nutrients and the focal organisms in order to formulate appropriate media recipes to support the metabolic needs. This step, which is often overlooked in proof-of-concept research, can substantially improve process metrics. After carbon, nitrogen is the most important and costly nutrient and should be given thorough consideration. In this study, we utilized nitrogen sourcing to tackle the biological upcycling of thermally oxo-degraded plastic waste using a previously described non-conventional yeast with the ability to utilize hydrophobic substrates, Candida maltosa. We compare the use of algae extract and casamino acids as organic, amino acid-based nitrogen sources to inorganic ammonium sulfate with the goal of increasing growth metric and biomass production. Our findings show that the use of algae extract and casamino acids promotes 2X–25X higher growth in model compounds and up to 2X on TOD products. Significant changes were observed in elemental composition of the cells in response to the change in nitrogen and carbon source. These changes align with the ~ 3X increase in internal protein content in the case of cells grown on casamino acids and TOD. Membrane properties and fatty acid content of the cells are also largely impacted, with a significant decrease in saturated and unsaturated fatty acid content, resulting in a reduced average lipid length in the case of casamino acid and TOD grown cells. Three proteins, namely the nonspecific lipid transfer protein POX18, the glycine zipper 2 transmembrane domain-containing protein, and the histidine triad nucleotide binding protein HNT1 were expressed in cells grown on TOD. The results from this study highlight the importance of nutrient management in non-model organisms and the biological challenges associated with the utilization of these compounds which can be insightful in future genetic engineering efforts for improved performance. The online version contains supplementary material available at 10.1186/s40643-026-01046-z.

Candidates:
A. The evidence does not state that however, research still lags on strategies to enable economic viability of these types of bioprocesses relative to the traditional production methods.
B. However, research still lags on strategies to enable economic viability of these types of bioprocesses relative to the traditional production methods.
C. The evidence does not state that bioprocesses grant us with broad opportunities to build a circular carbon economy.
D. Tuning the composition of the microbial growth medium is not one way to address these issues.
E. The evidence does not state that using microbes allows us to replace non-renewable resources and change waste management strategies, including recycling and upcycling.
F. Tuning the composition of the microbial growth medium is one way to address these issues.
G. Using microbes allows us to replace non-renewable resources and change waste management strategies, including recycling and upcycling.
H. Bioprocesses grant us with broad opportunities to build a circular carbon economy.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Unlike conventional monoclonal antibodies, complex biologics—such as bispecific antibodies and fusion proteins—often face challenges including lower expression levels, higher mispairing rates, and greater sensitivity to culture conditions, which can collectively limit both titer and product quality. Leveraging process development case studies, this review systematically explores upstream bioprocessing strategies aimed at mitigating these challenges, with a focus on titer enhancement, lactate metabolism regulation, acidic charge variants control, reduction of aggregates and fragments, and glycosylation optimization. Finally, a perspective toward future upstream development strategies is discussed.

Candidates:
A. Unlike conventional monoclonal antibodies, complex biologics—such as bispecific antibodies and fusion proteins—often face challenges including lower expression levels, higher mispairing rates, and greater sensitivity to culture conditions, which can collectively limit both titer and product quality.
B. Finally, a perspective toward future upstream development strategies is discussed.
C. Leveraging process development case studies, this review systematically explores upstream bioprocessing strategies aimed at mitigating these challenges, with a focus on titer enhancement, lactate metabolism regulation, acidic charge variants control, reduction of aggregates and fragments, and glycosylation optimization.
D. Finally, a perspective toward future upstream development strategies is not discussed.
E. Unlike conventional monoclonal antibodies, complex biologics—such as bispecific antibodies and fusion proteins—often face challenges including lower expression levels, higher mispairing rates, and greater sensitivity to culture conditions, which cannot collectively limit both titer and product quality.
F. The evidence does not state that leveraging process development case studies, this review systematically explores upstream bioprocessing strategies aimed at mitigating these challenges, with a focus on titer enhancement, lactate metabolism regulation, acidic charge variants control, reduction of aggregates and fragments, and glycosylation optimization.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
This study addresses the low natural yield of milbemycin D, a potent but underutilized insecticidal macrolide, by engineering a high-producing microbial strain. Using an optimized CRISPR/Cas9-AcrIIA4 system, we replaced the aveA3 polyketide synthase gene in Streptomyces avermitilis HU501 with the heterologous milA3 gene from Streptomyces bingchenggensis , effectively redirecting biosynthesis toward milbemycin D. Through fermentation optimization, a titer of 679.03 mg/L was achieved. Bioassays demonstrated that the biosynthesized milbemycin D exhibits enhanced activity against key pests compared to commercial milbemycin A3/A4. This work establishes a high-yield, laboratory-scale platform for milbemycin D production and highlights CRISPR/Cas9-driven combinatorial biosynthesis as a powerful tool for accessing high-value natural products.

Candidates:
A. Using an optimized CRISPR/Cas10-AcrIIA4 system, we replaced the aveA3 polyketide synthase gene in Streptomyces avermitilis HU501 with the heterologous milA3 gene from Streptomyces bingchenggensis , effectively redirecting biosynthesis toward milbemycin D.
B. Bioassays demonstrated that the biosynthesized milbemycin D exhibits enhanced activity against key pests compared to commercial milbemycin A4/A4.
C. Through fermentation optimization, a titer of 679.03 mg/L was achieved.
D. Using an optimized CRISPR/Cas9-AcrIIA4 system, we replaced the aveA3 polyketide synthase gene in Streptomyces avermitilis HU501 with the heterologous milA3 gene from Streptomyces bingchenggensis , effectively redirecting biosynthesis toward milbemycin D.
E. Bioassays demonstrated that the biosynthesized milbemycin D exhibits enhanced activity against key pests compared to commercial milbemycin A3/A4.
F. The evidence does not state that this study addresses the low natural yield of milbemycin D, a potent but underutilized insecticidal macrolide, by engineering a high-producing microbial strain.
G. This study addresses the low natural yield of milbemycin D, a potent but underutilized insecticidal macrolide, by engineering a high-producing microbial strain.
H. Through fermentation optimization, a titer of 680.03 mg/L was achieved.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
3‐Methyl‐1‐butanol (3‐MB), a promising next‐generation biofuel, has garnered significant interest owing to its superior combustion characteristics and fuel compatibility. However, current 3‐MB biosynthesis faces major challenges, including low production efficiency and severe toxicity‐induced growth inhibition, which significantly limit its industrial feasibility. In this study, we systematically developed an integrated metabolic engineering approach for high‐level 3‐MB production in Escherichia coli . Through semi‐rational engineering of the rate‐limiting enzyme dihydroxyacid dehydratase (DHAD), combined with molecular dynamics simulations, we identified and addressed previously unrecognized catalytic bottlenecks. The engineered strain exhibited a 32.3‐fold increase in 3‐MB production, reaching 2.20 g/L in shake‐flask cultures. Subsequent adaptive laboratory evolution further improved strain robustness, while genomic analysis revealed novel regulatory targets for metabolic optimization. In a scaled‐up bioreactor fermentation system, the final strain achieved a record titer of 6.24 g/L, representing the highest reported titer for engineered microbial systems. This work not only establishes a scalable platform for 3‐MB biosynthesis but also provides a modular engineering framework applicable to other advanced biofuels. Keywords: 3‐methyl‐1‐butanol, Escherichia coli , metabolic engineering, semi‐rational design, system optimization


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Mesenchymal stem cells (MSCs) are multipotent cells that have the ability to mediate cellular repair through a combination of soluble paracrine factors, as well as bioactive cargo packaged within extracellular vesicles (EVs). Although MSC-derived EVs have been widely investigated for their regenerative potential, progress toward translational evaluation has been limited in part by challenges in scalable and reproducible manufacturing. We recently reported that human telomerase reverse transcriptase (hTERT)-immortalized MSCs reproducibly produce EVs that retain key characteristics of EVs derived from primary MSCs. Building on this work, three-dimensional (3D) culture systems have emerged as promising platforms for large-scale manufacturing. In this study, we compared the yield, molecular composition, and functional activity of EVs produced from hTERT-immortalized MSCs cultured in either a fixed-bed bioreactor or conventional two-dimensional (2D) flasks. Our data demonstrate that bioreactor culture results in increased EV yield as compared to an equivalent production from 2D cultures. Molecular analyses indicated that bioreactor-derived EVs were associated with a broader spectrum of cargo and were enriched with molecules that may contribute to enhanced reparative function. Importantly, bioreactor-derived EVs also exerted a more pronounced effect in cellular repair assays in vitro. Collectively, these results highlight the potential of fixed-bed bioreactors as scalable platforms for EV production, offering higher yields while preserving molecular composition and functional activity. This approach represents an important step toward achieving the reproducible, high-quality EV production required for research and future translational applications.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Alternative substrates to traditional Camellia sinensis tea are increasingly investigated to diversify kombucha and enhance its functional properties. This review synthesizes evidence (2020–2025) on how non-tea substrates influence microbial ecology, metabolite composition, and bioactivity of kombucha. A semi-systematic search of PubMed, Scopus, Web of Science, and publisher platforms identified studies on fruit, vegetable, herbal, algal, cereal, dairy, and food-industry by-product substrates reporting compositional or functional outcomes. Extracted data included substrate characteristics, fermentation conditions, SCOBY features, analytical methods, and reported antioxidant, anti-inflammatory, metabolic, probiotic, and dermatological effects. Fermentation often leads to an increase in total phenolic content and antioxidant capacity. These effects are highly dependent on fermentation conditions, particularly duration and substrate composition. In some cases, prolonged fermentation may result in phenolic degradation or transformation, leading to reduced levels of certain compounds. Fruit- and hibiscus-based systems enhanced anthocyanin-driven antioxidant and anti-inflammatory activity. Vegetable and cereal substrates supplied phenolic acids and β -glucans associated with metabolic regulation and gut health, whereas by-products and algal fermentations supported waste valorization and enrichment in chlorogenic acids, pigments, fibers, and peptides. Despite promising functionality, substantial inter-study variability and limited in vivo validation and the lack of standardized fermentation protocols constrain translational application. In addition, the inherent variability in SCOBY microbial composition represents a major source of inconsistency, as differences in microbial communities can significantly influence fermentation dynamics, metabolite profiles, and functional outcomes.

Candidates:
A. A semi-systematic search of PubMed, Scopus, Web of Science, and publisher platforms identified studies on fruit, vegetable, herbal, algal, cereal, dairy, and food-industry by-product substrates reporting compositional or functional outcomes.
B. This review synthesizes evidence (2021–2025) on how non-tea substrates influence microbial ecology, metabolite composition, and bioactivity of kombucha.
C. The evidence does not state that extracted data included substrate characteristics, fermentation conditions, SCOBY features, analytical methods, and reported antioxidant, anti-inflammatory, metabolic, probiotic, and dermatological effects.
D. The evidence does not state that a semi-systematic search of PubMed, Scopus, Web of Science, and publisher platforms identified studies on fruit, vegetable, herbal, algal, cereal, dairy, and food-industry by-product substrates reporting compositional or functional outcomes.
E. Extracted data included substrate characteristics, fermentation conditions, SCOBY features, analytical methods, and reported antioxidant, anti-inflammatory, metabolic, probiotic, and dermatological effects.
F. Alternative substrates to traditional Camellia sinensis tea are not increasingly investigated to diversify kombucha and enhance its functional properties.
G. Alternative substrates to traditional Camellia sinensis tea are increasingly investigated to diversify kombucha and enhance its functional properties.
H. This review synthesizes evidence (2020–2025) on how non-tea substrates influence microbial ecology, metabolite composition, and bioactivity of kombucha.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Amines are indispensable structural motifs in pharmaceuticals, agrochemicals, functional materials, and beyond, driving continuous demand for efficient synthetic methods. While established strategies like cross-coupling and reductive amination are prevalent, traditional batch processes often suffer from limitations in mixing, heat/mass transfer, safety, and scalability. Flow chemistry emerges as a powerful process intensification technology, offering enhanced transport properties, precise parameter control, and improved safety profiles, thereby presenting a highly efficient approach for amine synthesis. This review systematically summarizes representative advances in flow chemistry for amination reactions from 2015 onward. It encompasses a broad range of enabling scenarios (e.g., heterogeneous, thermally activated, and enzymatic amination, among others), analyzed through the lens of process intensification. This review also examines the development of novel continuous-flow amination processes and the study of reaction kinetics leveraging flow chemistry. By providing a consolidated reference on the field’s evolution over the past decade, this review aims to guide researchers toward developing more efficient, sustainable, and scalable flow-based amination processes.

Candidates:
A. Amines are indispensable structural motifs in pharmaceuticals, agrochemicals, functional materials, and beyond, driving continuous demand for efficient synthetic methods.
B. This review systematically summarizes representative advances in flow chemistry for amination reactions from 2016 onward.
C. Flow chemistry emerges as a powerful process intensification technology, offering enhanced transport properties, precise parameter control, and improved safety profiles, thereby presenting a highly efficient approach for amine synthesis.
D. The evidence does not state that flow chemistry emerges as a powerful process intensification technology, offering enhanced transport properties, precise parameter control, and improved safety profiles, thereby presenting a highly efficient approach for amine synthesis.
E. While established strategies like cross-coupling and reductive amination are not prevalent, traditional batch processes often suffer from limitations in mixing, heat/mass transfer, safety, and scalability.
F. This review systematically summarizes representative advances in flow chemistry for amination reactions from 2015 onward.
G. While established strategies like cross-coupling and reductive amination are prevalent, traditional batch processes often suffer from limitations in mixing, heat/mass transfer, safety, and scalability.
H. Amines are not indispensable structural motifs in pharmaceuticals, agrochemicals, functional materials, and beyond, driving continuous demand for efficient synthetic methods.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The widespread utilization of synthetic dyes within the textile industry, driven by their chemical recalcitrance and diverse chromatic spectra, constitutes a significant global environmental challenge. Improper discharge of these highly stable effluents into natural water bodies leads to severe ecological imbalances, affecting aquatic life and soil integrity while posing indirect risks to human health due to their mutagenic potential. Conventional physicochemical treatment methods are often hindered by prohibitive operational costs and the frequent generation of hazardous secondary pollutants. Consequently, there is an urgent demand for sustainable biotechnological alternatives to mitigate these industrial impacts. Bioremediation, specifically using white-rot fungi, represents a robust and eco-friendly strategy for the degradation of complex aromatic structures. Species such as Trametes versicolor , Pleurotus ostreatus , and Phanerochaete chrysosporium utilize a specialized extracellular enzymatic complex to mineralize toxic compounds effectively. Here we review the ligninolytic capacity of white-rot fungi and their specialized enzymatic systems for environmental sustainability. The primary points are: (i) the biochemical mechanisms of the ligninolytic system of laccases and peroxidases during dye degradation; (ii) the influence of operational parameters such as pH, temperature, and nutrient availability on fungal metabolic efficiency; (iii) the diverse environmental applications of these microorganisms in treating real textile effluents; (iv) the current biotechnological challenges, including maintaining enzymatic stability in non-sterile industrial environments; and (v) the future perspectives for scaling up fungal treatment systems from laboratory research to large-scale industrial implementation.

Candidates:
A. Improper discharge of these highly stable effluents into natural water bodies leads to severe ecological imbalances, affecting aquatic life and soil integrity while posing indirect risks to human health due to their mutagenic potential.
B. The widespread utilization of synthetic dyes within the textile industry, driven by their chemical recalcitrance and diverse chromatic spectra, constitutes a significant global environmental challenge.
C. Conventional physicochemical treatment methods are not often hindered by prohibitive operational costs and the frequent generation of hazardous secondary pollutants.
D. Consequently, there is an urgent demand for sustainable biotechnological alternatives to mitigate these industrial impacts.
E. Conventional physicochemical treatment methods are often hindered by prohibitive operational costs and the frequent generation of hazardous secondary pollutants.
F. Consequently, there is not an urgent demand for sustainable biotechnological alternatives to mitigate these industrial impacts.
G. The evidence does not state that the widespread utilization of synthetic dyes within the textile industry, driven by their chemical recalcitrance and diverse chromatic spectra, constitutes a significant global environmental challenge.
H. The evidence does not state that improper discharge of these highly stable effluents into natural water bodies leads to severe ecological imbalances, affecting aquatic life and soil integrity while posing indirect risks to human health due to their mutagenic potential.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Hydrogen/deuterium exchange-mass spectrometry (HX-MS) is a rapidly expanding technique used to investigate protein conformational ensembles. The growing popularity and utility of HX-MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation. Most HX-MS data formats also employ only mean deuteration representations of the data rather than full isotopic mass spectra, which reduces the information content of the data and limits downstream quantitative analysis. Inspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX-MS data. The HXMS format preserves the isotopic mass envelopes for all peptides, captures the full experimental time-course including fully deuterated control samples, and contains all other key information. It supports multimodal distributions, post-translational modifications (PTMs), and experimental replicates. To promote compatibility with existing HX-MS workflows, we also developed PFLink, a Python package that converts exported data files from commonly used HX-MS software to the HXMS format. PFLink and the HXMS format will enable quantitative, higher-resolution data processing, improved data sharing and storage among HX-MS practitioners, future machine learning applications, and further developments in HX-MS analysis. PFLink is publicly available to install locally on HuggingFace, alongside documentation, or use online at HuggingFace ( https://huggingface.co/spaces/glasgow-lab/PFlink ). The supplementary information includes sample input files, sample HXMS files, and a generic unfilled PFlink custom CSV file that users may populate with key experimental conditions and results, which can then be read and converted into the HXMS format.

Candidates:
A. The evidence does not state that the growing popularity and utility of HX-MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation.
B. Hydrogen/deuterium exchange-mass spectrometry (HX-MS) is a rapidly expanding technique used to investigate protein conformational ensembles.
C. The evidence does not state that most HX-MS data formats also employ only mean deuteration representations of the data rather than full isotopic mass spectra, which reduces the information content of the data and limits downstream quantitative analysis.
D. The growing popularity and utility of HX-MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation.
E. Hydrogen/deuterium exchange-mass spectrometry (HX-MS) is not a rapidly expanding technique used to investigate protein conformational ensembles.
F. Inspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX-MS data.
G. The evidence does not state that inspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX-MS data.
H. Most HX-MS data formats also employ only mean deuteration representations of the data rather than full isotopic mass spectra, which reduces the information content of the data and limits downstream quantitative analysis.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
The magnetotactic bacterium Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe 3 O 4 ) nanocrystals, known as magnetosomes. Owing to their uniform size, purity and superior magnetic properties, magnetosomes represent highly attractive nanomaterials for biotechnological and biomedical applications. However, their bioproduction is limited by demanding cultivation requirements, largely because magnetite biomineralization is highly sensitive to environmental parameters, particularly oxygen. While elevated oxygen concentrations are known to inhibit magnetosome formation, quantitative analyses under defined low‐oxygen conditions are scarce. Here, we cultivated MSR‐1 in bioreactors under precisely controlled dissolved oxygen (DO) levels and quantified growth behaviour, substrate uptake and magnetosome characteristics. Cells harvested during late exponential growth revealed that magnetite crystal numbers per cell were similar across a wide DO range (0%–5%), whereas crystal sizes decreased with increasing oxygen levels. The data further indicate that oxygen inhibits biomineralization primarily through direct oxidative interference rather than indirect metabolic effects. These findings provide a mechanistic basis for optimizing oxygen control strategies in MTB cultivation and demonstrate that fine‐tuning DO levels enables targeted modulation of magnetosome size and properties. This advances both the bioprocess development of high‐yield magnetosome production and the application of tailored magnetic nanoparticles in biotechnology and medicine.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Tobacco aging is a critical for developing desirable flavor profiles, driven primarily by microbial and enzymatic activities. This study systematically evaluated the effects of Bacillus clausii inoculation (FJ) and B. clausii -cellulase co-treatment (JM) on the surface microbial communities and aroma compounds of Yunyan 87 tobacco leaves during 9 months of aging, with natural aging as the control (CK). High-throughput 16S rRNA sequencing revealed that the JM treatment significantly increased microbial diversity and enhanced the structural similarity of microbial communities across different aging stages compared with the CK and FJ treatments, while also promoting the proliferation of beneficial taxa (e.g., Bacteroidota , Cyanobacteria ). PICRUSt functional prediction revealed that JM enriched metabolic pathways related to carbohydrate metabolism (18.2–20.6%), amino acid metabolism (15.1–16.7%), and metabolism of cofactors and vitamins (16.5–17.7%)–key pathways for flavor precursor conversion. A total of 29 key aroma compounds (odor activity value > 1) were significantly influenced the quality of tobacco leaves, with JM significantly increasing total volatile content (317.1–388.8 μg/g vs. 124.7–143.0 μg/g in CK and 283.2–300.8 μg/g in FJ). Notably, JM promoted the accumulation of esters (ethyl palmitate, ethyl linoleate) and ketones (4,7,9-megastigmatrien-3-one, damascenone), contributing desirable fruity, floral, and sweet notes. Correlation analysis linked Pseudomonas , Bacillus , and Acinetobacter to the formation of key volatiles. These findings demonstrate that microbial-enzyme co-fermentation enriched desirable aromas, and stabilized microbial communities, providing a practical and efficient strategy for industrial production of high-quality aged tobacco. The online version contains supplementary material available at 10.1186/s40643-026-01052-1.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The transition to a forest-based economy relies on sustainable alternatives that can efficiently convert renewable resources into chemical and material products. Sugar-centered wood fractionation processes, also called modern biorefineries, offer significant potential as a renewable precursor for the substitution of petrochemical derivatives. However, unlocking the full potential of advanced biorefineries requires optimized pretreatment strategies to valorize all biomass-side streams. This work evaluated the distinctive and connective applications of nitrogen explosive decompression (NED), protic ionic liquid (PIL), and alkaline pretreatments for the production of monosaccharides, oligosaccharides, and high value lignin from aspen ( Populus tremula ) biomass within a biorefinery context. Each pretreatment method uniquely influenced biomass fractionation, affecting the hemicellulose and cellulose dissolution, delignification, and the physicochemical properties of the remaining cellulose and lignin components. The findings indicate that PIL pretreatment alone yielded 56% monomeric sugars upon saccharification. Additionally, the combinatorial PIL-NED and Alkaline-NED pretreatment was superior for lignin removal, achieving 68% and 85% delignification while concurrently generating valuable side streams. Henceforth, integrated PIL-NED collaborative approach presents a considerable cost and efficiency advantage over conventional biorefinery pretreatments, offering a promising pathway for the co-production of monosaccharides and high-quality lignin. The online version contains supplementary material available at 10.1186/s40643-026-01040-5.

Candidates:
A. The evidence does not state that sugar-centered wood fractionation processes, also called modern biorefineries, offer significant potential as a renewable precursor for the substitution of petrochemical derivatives.
B. The evidence does not state that however, unlocking the full potential of advanced biorefineries requires optimized pretreatment strategies to valorize all biomass-side streams.
C. Sugar-centered wood fractionation processes, also called modern biorefineries, offer significant potential as a renewable precursor for the substitution of petrochemical derivatives.
D. The evidence does not state that this work evaluated the distinctive and connective applications of nitrogen explosive decompression (NED), protic ionic liquid (PIL), and alkaline pretreatments for the production of monosaccharides, oligosaccharides, and high value lignin from aspen ( Populus tremula ) biomass within a biorefinery context.
E. This work evaluated the distinctive and connective applications of nitrogen explosive decompression (NED), protic ionic liquid (PIL), and alkaline pretreatments for the production of monosaccharides, oligosaccharides, and high value lignin from aspen ( Populus tremula ) biomass within a biorefinery context.
F. However, unlocking the full potential of advanced biorefineries requires optimized pretreatment strategies to valorize all biomass-side streams.
G. The transition to a forest-based economy relies on sustainable alternatives that can efficiently convert renewable resources into chemical and material products.
H. The transition to a forest-based economy relies on sustainable alternatives that cannot efficiently convert renewable resources into chemical and material products.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["46.9%", "47.9%"]

Evidence:
Diabetes, particularly type 2 diabetes mellitus (T2DM), has emerged as a major global health challenge, while currently available therapeutic drugs are frequently associated with drug resistance and adverse side effects. In recent years, bioactive peptides have attracted increasing attention as promising hypoglycaemic candidates due to their favorable safety profiles, good tolerability, and multi‐target physiological regulatory activities. As a GRAS‐designated unicellular green alga, Chlamydomonas reinhardtii is characterized by a high protein content, with approximately 46.9% of its dry biomass composed of protein, making it a rich source for the development of hypoglycaemic short peptides. Previous studies have shown that proteolytic products derived from C. reinhardtii markedly inhibit key glucose‐regulating enzymes, including α‐amylase and α‐glucosidase, exhibiting superior inhibitory potential compared with various terrestrial protein sources. Nevertheless, challenges remain in the large‐scale production of C. reinhardtii ‐derived hypoglycaemic peptides, precise control of the proteolytic process, and validation of their in vivo efficacy. This review summarizes the major sources of hypoglycaemic peptides, current extraction and characterization strategies, and their underlying mechanisms of action, while highlighting the application potential and key limitations of C. reinhardtii ‐derived hypoglycaemic peptides. Addressing these challenges is expected to facilitate the development and application of C. reinhardtii ‐based functional foods and nutraceuticals for diabetes management.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Advances in live-cell fluorescence microscopy have enabled us to visualize single molecules (such as mRNAs and nascent proteins) in real time with high spatiotemporal resolution. However, these experiments generate large datasets that require complex computational processing pipelines to derive meaningful and quantitative information, which is a technical barrier for many researchers. Here, we introduce MicroLive, an open-source Python-based application for quantifying live-cell microscopy images. MicroLive provides an interactive Graphical User Interface (GUI) to perform key tasks, including cell segmentation, photobleaching correction, single-particle detection/tracking, spot intensity quantification, inter-channel colocalization, and time-series correlation analysis. As a ground-truth testing dataset, we used synthetic live-cell imaging data generated with the rSNAPed toolkit, demonstrating accurate extraction of biologically relevant parameters. Microscopy images of U-2 OS cells expressing a gene construct smHA-KDM5B-BoxB-MS2 were used to demonstrate the use of this software. MicroLive is distributed under a GPLv3 license and available on GitHub https://github.com/ningzhaoAnschutz/microlive . It can be installed via pip: pip install microlive .

Candidates:
A. MicroLive provides an interactive Graphical User Interface (GUI) to perform key tasks, including cell segmentation, photobleaching correction, single-particle detection/tracking, spot intensity quantification, inter-channel colocalization, and time-series correlation analysis.
B. However, these experiments generate large datasets that require complex computational processing pipelines to derive meaningful and quantitative information, which is not a technical barrier for many researchers.
C. Here, we introduce MicroLive, an open-source Python-based application for quantifying live-cell microscopy images.
D. The evidence does not state that advances in live-cell fluorescence microscopy have enabled us to visualize single molecules (such as mRNAs and nascent proteins) in real time with high spatiotemporal resolution.
E. The evidence does not state that here, we introduce MicroLive, an open-source Python-based application for quantifying live-cell microscopy images.
F. The evidence does not state that microLive provides an interactive Graphical User Interface (GUI) to perform key tasks, including cell segmentation, photobleaching correction, single-particle detection/tracking, spot intensity quantification, inter-channel colocalization, and time-series correlation analysis.
G. However, these experiments generate large datasets that require complex computational processing pipelines to derive meaningful and quantitative information, which is a technical barrier for many researchers.
H. Advances in live-cell fluorescence microscopy have enabled us to visualize single molecules (such as mRNAs and nascent proteins) in real time with high spatiotemporal resolution.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["621 kPa", "100%", "620 kPa", "2 h", "99%", "1 h", "89%", "88%"]

Evidence:
Photosynthetic microorganisms are capable of oxygenic photosynthesis, delivering both oxygen and cofactors to drive enzymatic redox reactions. However, their dependence on visible light limits the tolerable cell densities to achieve high reaction rates. Immobilizing cells within a matrix often increases biocatalyst productivity while allowing facile retainment but also creates mass transfer limitations across the solid–liquid interface. Herein, we address these challenges and present the immobilization of recombinant cyanobacteria in 3D-printed hydrogels of varying geometries. In particular, whole cells of the cyanobacterium Synechocystis sp. PCC 6803, engineered to express the gene of the ene-reductase YqjM, were implemented in biocompatible hydrogels made out of nanofibrillated cellulose and alginate. The hydrogels were 3D-printed via extrusion into different geometries to alleviate light and mass transfer limitations and were applied for the reduction of prochiral 2-methylmaleimide to ( R )-2-methylsuccinimide. The obtained reactors exhibit high mechanical stability (620 kPa), efficient flow and mass transfer characteristics, high specific surface area (up to 2129 mm 2 g –1 ), and retention times favorable to achieve high product formation. ( R )-2-methylsuccinimide was obtained with a space–time yield of 0.28 g L –1 h –1 and a high enantiomeric purity (>99%). The highly atom-efficient chemical process (88%) using only water to provide electrons for NADPH regeneration could be upscaled and can potentially be operated in extended periods to reduce wastewater associated with cell cultivation. Overall, 3D printing of photosynthetic microorganisms embedded in a hydrogel matrix holds significant promise for advancing the development of whole-cell solid-state photosynthetic cell factories. These are important steps toward improved reactor designs and higher efficiencies to improve crucial redox biotransformations.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Natural killer cells are emerging as promising “off-the-shelf” effectors for cancer immunotherapy, yet expansion of the NK-92 cell line in batch cultivation leads to rapid loss of cytotoxicity concomitant with lactate accumulation. In this study we developed and validated a two-phase manufacturing strategy that decouples cell proliferation from functional recovery in order to obtain an improved final product potency. Our 8-day kinetic survey determined declines in viability, metabolite profiles and cytotoxicity during static batch expansion. Guided by these data, in a 32-run full-factorial design-of-experiments approach we varied fresh cultivation medium proportion, temperature, dissolved oxygen, and recovery duration; partial least squares modeling identified fresh-medium ratio and recovery time as the primary drivers of cytotoxicity restoration. Identified optimal conditions (90% fresh medium, 37.2 °C, 3.7 days recovery) recovered cytotoxicity and maximized cytotoxic capacity. These setpoints were then translated to a 2 L stirred-tank bioreactor, where a fed-batch expansion under controlled pH and lactate levels produced 2.0 × 10 9 cells, followed by recovery that achieved 43% ± 8% cytotoxicity. This scalable, two-phase paradigm minimizes medium usage and obviates continuous perfusion, offering a potential workflow to increase NK-92 potency and a base for manufacturing high-quality advanced therapy medicinal products.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Lacticaseibacillus rhamnosus is widely studied for strain‐specific antimicrobial and immunomodulatory properties, but comparative data for viable and heat‐inactivated preparations remain limited. This study compared the anti‐pathogenic activity of live L. rhamnosus IDCC 3201 and its commercially prepared heat‐inactivated formulation, RHT 3201, and examined their associated microbiota and metabolite profiles in a single‐donor ex vivo fecal fermentation model. Transcript‐level immunomodulatory activity was evaluated separately for RHT 3201 in LPS‐stimulated RAW 264.7 cells. Both preparations inhibited key intestinal pathogens, including Escherichia coli , Staphylococcus aureus , Enterococcus faecalis , and Salmonella Typhimurium, although live IDCC 3201 showed stronger inhibition against E. faecalis and S. Typhimurium . In LPS‐stimulated RAW 264.7 cells, RHT 3201 reduced IL‐1β, IL‐6, COX‐2, and TNF‐α mRNA expression by approximately 39%, 23%, 17%, and 16%, respectively, at 10 × 10 7 CFU/mL. At the species level in the ex vivo fermentation model, RHT 3201 was associated with a higher relative abundance of Bifidobacterium pseudocatenulatum and Lactobacillus ruminis , whereas L. rhamnosus was detected only in the IDCC 3201 group during the 24‐h culture period. Metabolite profiling also showed distinct product‐associated signatures between the two preparations, with higher lactic acid detected in RHT 3201. Overall, the two preparations showed comparable activity against some pathogens but distinct microbiota‐ and metabolite‐associated profiles in this single‐donor ex vivo system, while anti‐inflammatory activity was assessed only for RHT 3201 at the mRNA level. Additional multi‐donor, protein‐level, and in vivo studies are required to confirm the reproducibility and physiological relevance of these findings. Keywords: anti‐pathogenic, ex vivo fermentation, heat‐inactivated, Lacticaseibacillus rhamnosus , metabolite profiling, microbiota


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["31 °C", "28.3 °C", "38 °C", "30 °C", "15%", "16%", "37 °C", "27.3 °C"]

Evidence:
Enzymatic depolymerization of polyethylene terephthalate (PET), the world’s most widely used polyester, in seawater at ambient temperature offers a promising and energy-efficient route for freshwater-free plastic recycling. While a number of PET hydrolases have been reported in recent years, their potential under saline conditions remains largely unexplored. Here, we screened eight enzymes in artificial seawater at 30 °C and engineered the most active one, Is PETase, using a semi-rational strategy focused on rigidifying flexible sites. The resulting variant M8 showed simultaneous enhancementsin thermostability (ΔT m = + 27.3 °C), activity (1.14-fold increase) and soluble expression yield (14.3-fold increase). The overall depolymerization efficiency of M8 surpassed that of the thermostable benchmark enzymes DuraPETase and LCC-ICCG by 32.2- and 10.4-fold, respectively. Notably, M8 achieved continuous and efficient depolymerization of 15% (w/v) PET powder in natural seawater at 37 °C, yielding monomers at a rate of 15.4 mM/day, a concentration sufficient to support downstream bacterial assimilation. This work provides an efficient enzymatic platform and paves the way for fully integrated, seawater-based plastic bioconversion processes. The online version contains supplementary material available at 10.1007/s44307-026-00104-z.