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:
Polyphenols are structurally diverse plant secondary metabolites with broad biological activities and growing applications across the food, health, and materials sectors. Conventional extraction based on organic solvents (e.g., methanol, ethanol) is often energy-intensive, inefficient, and environmentally burdensome. Ionic liquids (ILs) and deep eutectic solvents (DESs) have therefore emerged as greener alternatives for polyphenol extraction. This review evaluates recent advances in solvent design, extraction performance, and process sustainability. Imidazolium-based ILs frequently achieve high yields and selectivity, particularly when coupled with ultrasound or microwave-assisted extraction, but high cost, synthetic complexity, viscosity-related constraints, and potential toxicity hinder scaleup. By contrast, DESs—especially those derived from choline chloride or lactic acid—are easier to prepare, less costly, and more compatible with industrial implementation, with efficiency enhanced by tailoring hydrogen bond networks, water content, and process intensification. Critical downstream challenges persist for both solvent classes, notably in extract purification and solvent recovery due to low volatility; approaches such as resin adsorption, antisolvent precipitation, and direct formulation have been explored. Overall, ILs and DESs represent compelling alternatives to conventional solvents, and future progress will depend on integrated extraction–recovery strategies, systematic solvent selection, and validation under scalable, sustainable processing conditions.


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: ["0.3%", "1.3%"]

Evidence:
Passiflora edulis (passion fruit) seed waste, an abundant by-product of the juice industry, is a promising source of piceatannol (PIC), a hydroxystilbene with superior antioxidant activity compared to resveratrol. However, its translation into a skin-targeted ingredient remains hindered by a lack of standardization and clinical validation. This review synthesizes current evidence on the dermatological potential of PIC and proposes a translational roadmap within a circular bioeconomy framework. Preclinical studies demonstrate that PIC exerts multi-target effects relevant to skin aging and acne, including ROS scavenging, anti-inflammatory activity via NF-κB/MAPK inhibition, suppression of melanogenesis, enhancement of hyaluronic acid and collagen synthesis, and antibacterial action against Cutibacterium acnes . However, clinical data are limited and methodologically inconsistent. To bridge this translational gap, we propose a development strategy focused on: (i) extract standardization with a proposed minimum PIC content (e.g., ≥0.3% w / w ); (ii) an integrated biorefinery approach for the co-production of seed oil and phenolic fractions; and (iii) a phase-gate pipeline encompassing dermal safety assessment, advanced delivery optimization, and biomarker-correlated clinical trials.


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:
Manure-rearing insects offer a sustainable protein source for feed, but managing microbial risks is crucial. To identify an optimal harvest stage balancing yield and safety, we tracked internal bacterial communities in two fly species across development. Bacterial communities shifted significantly, with specific taxa dominating later pupal and adult stages. In contrast, dispersing-stage larvae—which stop feeding and leave the manure—maintained a more stable bacterial profile and were easier to collect. Harvesting at this dispersing stage therefore provides a practical balance between biomass production and microbial safety, supporting the design of safer manure-to-feed systems.

Candidates:
A. Manure-rearing insects offer a sustainable protein source for feed, but managing microbial risks is crucial.
B. Bacterial communities shifted significantly, with specific taxa dominating later pupal and adult stages.
C. The evidence does not state that to identify an optimal harvest stage balancing yield and safety, we tracked internal bacterial communities in two fly species across development.
D. To identify an optimal harvest stage balancing yield and safety, we tracked internal bacterial communities in two fly species across development.
E. In contrast, dispersing-stage larvae—which stop feeding and leave the manure—maintained a more stable bacterial profile and were not easier to collect.
F. The evidence does not state that bacterial communities shifted significantly, with specific taxa dominating later pupal and adult stages.
G. Manure-rearing insects offer a sustainable protein source for feed, but managing microbial risks is not crucial.
H. In contrast, dispersing-stage larvae—which stop feeding and leave the manure—maintained a more stable bacterial profile and were easier to collect.


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:
Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae , a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae . We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1 , a Sec1/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor–mediated vesicle trafficking, which, in combination with SPAoalp1 , increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine κ-casein increased secretion from 0.11 to 0.24 mg/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC 90 values of 4.56–8.24% ( v / v ) against two Candida albicans strains and 4.68% ( v / v ) against Aspergillus niger . The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production.


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:
Chronic wounds represent a significant global healthcare challenge, affecting millions of patients and imposing substantial economic burdens on healthcare systems. Traditional wound management approaches often fail to address the complex pathophysiology underlying chronic wounds, including persistent inflammation, impaired angiogenesis, and disrupted extracellular matrix remodeling. Three-dimensional (3D) bioprinting has emerged as a transformative technology that enables the fabrication of patient-specific, biomimetic tissue constructs capable of addressing these intricate challenges. This comprehensive review synthesizes recent advances in 3D bioprinting for chronic wound treatment, examining bioprinting technologies, biomaterial innovations, mechanisms of wound healing, and clinical applications. Recent studies demonstrate that bioprinted constructs incorporating living cells, growth factors, and bioactive molecules can significantly accelerate wound closure, enhance vascularization, and restore functional skin architecture. Notable innovations include in situ bioprinting systems, photosynthetic scaffolds for oxygen delivery, and immunomodulatory bioinks. While significant technical challenges remain—including vascularization, scalability, and regulatory approval—the integration of advanced bioprinting techniques with regenerative medicine principles offers unprecedented opportunities for personalized chronic wound care and improved patient outcomes.

Candidates:
A. Traditional wound management approaches often fail to address the complex pathophysiology underlying chronic wounds, including persistent inflammation, impaired angiogenesis, and disrupted extracellular matrix remodeling.
B. This comprehensive review synthesizes recent advances in 3D bioprinting for chronic wound treatment, examining bioprinting technologies, biomaterial innovations, mechanisms of wound healing, and clinical applications.
C. The evidence does not state that chronic wounds represent a significant global healthcare challenge, affecting millions of patients and imposing substantial economic burdens on healthcare systems.
D. Chronic wounds represent a significant global healthcare challenge, affecting millions of patients and imposing substantial economic burdens on healthcare systems.
E. Three-dimensional (3D) bioprinting has emerged as a transformative technology that enables the fabrication of patient-specific, biomimetic tissue constructs capable of addressing these intricate challenges.
F. The evidence does not state that traditional wound management approaches often fail to address the complex pathophysiology underlying chronic wounds, including persistent inflammation, impaired angiogenesis, and disrupted extracellular matrix remodeling.
G. Three-dimensional (4D) bioprinting has emerged as a transformative technology that enables the fabrication of patient-specific, biomimetic tissue constructs capable of addressing these intricate challenges.
H. This comprehensive review synthesizes recent advances in 4D bioprinting for chronic wound treatment, examining bioprinting technologies, biomaterial innovations, mechanisms of wound healing, and clinical applications.


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: ["20%", "56.2%", "55.2%", "21%"]

Evidence:
Docosahexaenoic acid (DHA) is an essential ω-3 polyunsaturated fatty acid (PUFA) with high nutritional and pharmaceutical value. The marine protist Aurantiochytrium is a promising industrial DHA producer; however, its DHA biosynthesis via the PUFA synthase pathway co-produces ω-6 docosapentaenoic acid (DPA), limiting DHA purity. Here, we introduced an ω-3 desaturase from Phytophthora infestans (Pin-O3D) into Aurantiochytrium sp. SD116. Functional validation in an Escherichia coli system co-expressing the native PUFA synthase confirmed that Pin-O3D converts DPA to DHA, shifting the DHA/DPA ratio from 1:1 to 2:1. Pin-O3D was then integrated into the fatty acid synthase (FAS) locus, simultaneously attenuating FAS activity and enabling heterologous gene expression. The engineered strain ΔFAS-Pin-O3D exhibited significantly ( p < 0.0001 in t -test) increased DHA content (55.2% of total fatty acids) and DHA/DPA ratio (5.91) in shake flasks, with no negative impact on biomass or lipid accumulation. Fed-batch fermentation confirmed the scalability of this strategy, achieving a >20% increase in DHA/DPA ratio. This study demonstrates that combining heterologous ω-3 desaturase expression with FAS attenuation is an effective approach for optimizing PUFA profiles in Aurantiochytrium .


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:
Microalgae are increasingly recognized as renewable biofactories for producing high-value bioactive molecules. However, their industrial exploitation is limited by their rigid cell walls, metabolite heterogeneity, and the energy-intensive nature of the extraction processes. Recent advances in process-intensification technologies, including microwave-assisted, ultrasound-assisted, enzymatic, pressurized liquid, and supercritical CO 2 -based methods, have significantly improved extraction efficiency and selectivity, with reported lipid recoveries exceeding 40–50% in some microalgal systems and carotenoid recoveries approaching 90% under optimized conditions. NADES-assisted systems further enhance mass transfer and solubilization through tailored hydrogen-bonding interactions, enabling selective extraction of polar and semi-polar metabolites under mild conditions. However, limitations remain, including high viscosity, variability in extraction performance, and challenges in solvent recovery and scale-up. This review critically evaluates the extraction efficiency, mechanistic basis, and sustainability of NADES-assisted processes, highlighting key limitations and identifying research priorities for their integration into scalable microalgal biorefinery systems.

Candidates:
A. However, their industrial exploitation is not limited by their rigid cell walls, metabolite heterogeneity, and the energy-intensive nature of the extraction processes.
B. The evidence does not state that nADES-assisted systems further enhance mass transfer and solubilization through tailored hydrogen-bonding interactions, enabling selective extraction of polar and semi-polar metabolites under mild conditions.
C. NADES-assisted systems further enhance mass transfer and solubilization through tailored hydrogen-bonding interactions, enabling selective extraction of polar and semi-polar metabolites under mild conditions.
D. Recent advances in process-intensification technologies, including microwave-assisted, ultrasound-assisted, enzymatic, pressurized liquid, and supercritical CO 2 -based methods, have significantly improved extraction efficiency and selectivity, with reported lipid recoveries exceeding 40–50% in some microalgal systems and carotenoid recoveries approaching 90% under optimized conditions.
E. Recent advances in process-intensification technologies, including microwave-assisted, ultrasound-assisted, enzymatic, pressurized liquid, and supercritical CO 3 -based methods, have significantly improved extraction efficiency and selectivity, with reported lipid recoveries exceeding 40–50% in some microalgal systems and carotenoid recoveries approaching 90% under optimized conditions.
F. Microalgae are not increasingly recognized as renewable biofactories for producing high-value bioactive molecules.
G. However, their industrial exploitation is limited by their rigid cell walls, metabolite heterogeneity, and the energy-intensive nature of the extraction processes.
H. Microalgae are increasingly recognized as renewable biofactories for producing high-value bioactive molecules.


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:
Sustainable nanotechnologies derived from renewable resources are increasingly being positioned at the interface of green chemistry, advanced drug delivery, and translational pharmaceutics. Over the past decade, lignocellulosic nanomaterials, chitin/chitosan platforms, polysaccharide-based nanogels and nano-enabled hydrogels, lignin- and polyphenol-derived nanostructures, and bio-based lipid nanocarriers have been engineered through progressively eco-efficient routes, including solvent-minimized self-assembly, nanoprecipitation, spray drying, hot-melt extrusion, and microfluidic-assisted fabrication. This work provides a structured evidence map of nano-enabled drug delivery and therapeutic platforms derived from renewable biological resources. Specifically, we aim to (i) identify and classify nanoplatform classes and renewable feedstocks; (ii) summarize reported pharmaceutical critical quality attributes (CQAs) and performance and safety endpoints; and (iii) appraise how “renewability” and “green” claims are evidenced (feedstock origin vs. process sustainability) and how frequently translational readiness factors (scalability, quality control, regulatory alignment) are addressed. We critically compare renewable and conventional nanomaterial platforms across key translational dimensions, including carbon footprint, batch consistency, biodegradability, functional tunability, safety/persistence, and scale-up maturity. Finally, we delineate a practical translational pathway—from biomass sourcing and fractionation to nanoformulation, characterization/stability, and GMP scale-up—highlighting cross-cutting enablers such as lifecycle assessment, EHS/toxicology risk assessment, quality-by-design, and regulatory alignment. Collectively, the evidence supports renewable nanomaterials as viable, scalable candidates for next-generation therapeutics, provided that variability control, standardized characterization, and safety-by-design principles are embedded early in development.

Candidates:
A. The evidence does not state that this work provides a structured evidence map of nano-enabled drug delivery and therapeutic platforms derived from renewable biological resources.
B. This work provides a structured evidence map of nano-enabled drug delivery and therapeutic platforms derived from renewable biological resources.
C. Sustainable nanotechnologies derived from renewable resources are not increasingly being positioned at the interface of green chemistry, advanced drug delivery, and translational pharmaceutics.
D. Specifically, we aim to (i) identify and classify nanoplatform classes and renewable feedstocks; (ii) summarize reported pharmaceutical critical quality attributes (CQAs) and performance and safety endpoints; and (iii) appraise how “renewability” and “green” claims are not evidenced (feedstock origin vs.
E. Sustainable nanotechnologies derived from renewable resources are increasingly being positioned at the interface of green chemistry, advanced drug delivery, and translational pharmaceutics.
F. Specifically, we aim to (i) identify and classify nanoplatform classes and renewable feedstocks; (ii) summarize reported pharmaceutical critical quality attributes (CQAs) and performance and safety endpoints; and (iii) appraise how “renewability” and “green” claims are evidenced (feedstock origin vs.
G. Over the past decade, lignocellulosic nanomaterials, chitin/chitosan platforms, polysaccharide-based nanogels and nano-enabled hydrogels, lignin- and polyphenol-derived nanostructures, and bio-based lipid nanocarriers have been engineered through progressively eco-efficient routes, including solvent-minimized self-assembly, nanoprecipitation, spray drying, hot-melt extrusion, and microfluidic-assisted fabrication.
H. The evidence does not state that over the past decade, lignocellulosic nanomaterials, chitin/chitosan platforms, polysaccharide-based nanogels and nano-enabled hydrogels, lignin- and polyphenol-derived nanostructures, and bio-based lipid nanocarriers have been engineered through progressively eco-efficient routes, including solvent-minimized self-assembly, nanoprecipitation, spray drying, hot-melt extrusion, and microfluidic-assisted fabrication.


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:
Microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity. However, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds. While conventional techniques such as selective media and enrichment culture remain foundational, they are inherently limited by community biases and the inability to support low-abundance, oligotrophic species. To address these bottlenecks, a diverse array of innovative isolation strategies has emerged. This review systematically categorizes and evaluates these methodologies, ranging from in situ cultivation to high-resolution single-cell manipulation. We first examine membrane diffusion-based cultivation (e.g., iChip), which mimics natural microenvironments to resuscitate recalcitrant microbes. Subsequently, we explore high-throughput single-cell technologies, including microfluidics for physicochemical separation, optical tweezers for precise manipulation, and fluorescence-activated cell sorting (FACS). Special attention is given to Raman-activated cell sorting (RACS) as a label-free functional screening tool and reverse genomics for targeted capture. By synthesizing the strengths and limitations of these approaches, we propose integrated workflows designed to accelerate the mining of untapped microbial resources.

Candidates:
A. The evidence does not state that to address these bottlenecks, a diverse array of innovative isolation strategies has emerged.
B. However, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds.
C. To address these bottlenecks, a diverse array of innovative isolation strategies has emerged.
D. Microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity.
E. While conventional techniques such as selective media and enrichment culture remain foundational, they are inherently limited by community biases and the inability to support low-abundance, oligotrophic species.
F. While conventional techniques such as selective media and enrichment culture remain foundational, they are not inherently limited by community biases and the inability to support low-abundance, oligotrophic species.
G. The evidence does not state that microorganisms represent the Earth’s most abundant biomass and a vast reservoir of genetic diversity.
H. The evidence does not state that however, traditional agar plate methods fail to recover the vast majority of these species, leaving a “microbial dark matter” that holds immense potential for the discovery of novel antibiotics and bioactive compounds.


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:
As the primary threat to the global pig industry, ASFV is a giant DNA virus encoding numerous proteins that are of unknown function yet essential for viral replication and virulence. Here, we developed two monoclonal antibodies targeting the replication-essential protein pE120R and mapped its linear epitopes. Notably, these epitopes are surface-accessible and highly conserved among ASFV strains. This work offers potent tools for investigating pE120R’s role in the ASFV life cycle and contributes to a deeper understanding of viral pathogenesis.

Candidates:
A. This work offers potent tools for investigating pE121R’s role in the ASFV life cycle and contributes to a deeper understanding of viral pathogenesis.
B. Here, we developed two monoclonal antibodies targeting the replication-essential protein pE120R and mapped its linear epitopes.
C. As the primary threat to the global pig industry, ASFV is a giant DNA virus encoding numerous proteins that are of unknown function yet essential for viral replication and virulence.
D. Notably, these epitopes are surface-accessible and highly conserved among ASFV strains.
E. As the primary threat to the global pig industry, ASFV is not a giant DNA virus encoding numerous proteins that are of unknown function yet essential for viral replication and virulence.
F. Here, we developed two monoclonal antibodies targeting the replication-essential protein pE121R and mapped its linear epitopes.
G. Notably, these epitopes are not surface-accessible and highly conserved among ASFV strains.
H. This work offers potent tools for investigating pE120R’s role in the ASFV life cycle and contributes to a deeper understanding of viral pathogenesis.


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: ["252 g/L", "10 g/L", "11 g/L", "93%", "253 g/L", "94%", "201 g/L", "200 g/L"]

Evidence:
Trehalose 6-phosphate (T6P), often referred to as plant insulin, serves as a central signaling molecule that regulates carbon partitioning and sucrose flux in plants, thereby influencing key agronomic traits, such as grain yield and drought resilience. Foliar spraying of T6P has been shown to significantly enhance yield and stress tolerance of numerous grains and vegetables. To circumvent the dependency on coenzymes in natural T6P synthesis, we designed and validated an in vitro new-to-nature, coenzyme-free, minimal enzymatic pathway for the biosynthesis of T6P from maltose and polyphosphate. This three-enzyme cocktail contained maltose phosphorylase, trehalose 6-phosphate phosphorylase, and polyphosphate glucokinase, and did not involve any costly coenzymes, such as ATP or UDP. Through systematic optimization of experimental parameters (including pH, temperature, Mg²⁺, phosphate concentration, and enzyme ratios), a 93% molar yield of T6P was achieved from 10 g/L maltose. The scale-up of this in vitro bioprocess to a 100-mL bioreactor with 200 g/L maltose enabled the production of up to 541 mM T6P (i.e., 252 g/L T6P disodium salt) within two hours, corresponding to a very high volumetric productivity of 126 g/L/h. This study established a scalable and cost-competitive in vitro biomanufacturing of T6P. Chemical intervention based on timed foliar spraying of T6P to cultivated crops offers a simpler and safer agricultural practice compared to genetic modification of crops. The online version contains supplementary material available at 10.1186/s40643-026-01057-w. Keywords: Chemical intervention, Food security, In vitro BioTransformation, In vitro synthetic biology, Multi-enzyme molecular machine, Trehalose-6-phosphate


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:
Current therapies for prostate cancer are limited by toxicity and acquired resistance, motivating development of biocompatible nanotherapeutics. Here, Solanum nigrum L.-derived nanovesicles (SDNVs) were isolated and characterized, showing a mean diameter of 93.4 ± 0.2 nm and a particle concentration of (4.2 ± 0.4) × 10 11 /mL. SDNV uptake, antitumor activity, and safety were assessed in PC-3 and RWPE-1 cells, while efficacy and biodistribution were further evaluated in a subcutaneous PC-3 xenograft mouse model following oral administration. SDNVs were readily internalized by PC-3 cells and suppressed viability, proliferation, and migration while inducing apoptosis. Senescence and cytotoxicity were not observed in RWPE-1 cells, indicating tumor-selective activity. The underlying mechanism was further investigated by transcriptome sequencing, followed by quantitative PCR, Western blotting, and pharmacologic rescue with the p53 inhibitor pifithrin-α (PFT-α). Transcriptomic analysis revealed 5058 differentially expressed genes, and further indicated activation of the p53/p21 axis and promoted cellular senescence in PC-3 cells after SDNV treatment. In vivo, oral SDNVs significantly reduced xenograft growth and showed no evident histopathologic toxicity in major organs. Collectively, SDNVs represent a plant-derived, biocompatible nanotherapeutic platform that selectively restrains prostate cancer growth via p53/p21-mediated senescence, supporting further development of senescence-targeting SDNV-based interventions. The online version contains supplementary material available at 10.1186/s40643-026-01055-y. Keywords: Plant-derived nanovesicles, Senescence, Prostate cancer, P53, Herbal medicine

Candidates:
A. SDNVs were readily internalized by PC-4 cells and suppressed viability, proliferation, and migration while inducing apoptosis.
B. SDNV uptake, antitumor activity, and safety were assessed in PC-3 and RWPE-1 cells, while efficacy and biodistribution were further evaluated in a subcutaneous PC-3 xenograft mouse model following oral administration.
C. SDNV uptake, antitumor activity, and safety were assessed in PC-4 and RWPE-1 cells, while efficacy and biodistribution were further evaluated in a subcutaneous PC-3 xenograft mouse model following oral administration.
D. Current therapies for prostate cancer are limited by toxicity and acquired resistance, motivating development of biocompatible nanotherapeutics.
E. Here, Solanum nigrum L.-derived nanovesicles (SDNVs) were isolated and characterized, showing a mean diameter of 93.4 ± 0.2 nm and a particle concentration of (4.2 ± 0.4) × 10 11 /mL.
F. Current therapies for prostate cancer are not limited by toxicity and acquired resistance, motivating development of biocompatible nanotherapeutics.
G. SDNVs were readily internalized by PC-3 cells and suppressed viability, proliferation, and migration while inducing apoptosis.
H. Here, Solanum nigrum L.-derived nanovesicles (SDNVs) were isolated and characterized, showing a mean diameter of 94.4 ± 0.2 nm and a particle concentration of (4.2 ± 0.4) × 10 11 /mL.


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:
Scalable suspension culture technologies are essential for the large-scale manufacturing of mesenchymal stem/stromal cells (MSCs). However, conventional microcarrier-based systems often fail to achieve scalable efficiency because cell–microcarrier dynamics vary across scales, necessitating complex, scale-dependent agitation protocols. To overcome this limitation, we designed a scale-up-oriented suspension culture system employing fluffy, fibrillated nanofiber scaffolds composed of chitosan and chitin. These nanofibers were readily suspended under continuous, gentle agitation and trapped cells on their surfaces, while spontaneously forming fluffy cell–scaffold aggregates through scaffold agglomeration. The low-adhesive chitosan nanofibers acted as physical spacers that prevented aggregate coalescence, thereby maintaining a microenvironment favorable for proliferation. By defining cell–scaffold aggregate size as the key scaling parameter—a biology-centric approach—, we successfully achieved scale-up from 30 mL to 5 L under continuous gentle agitation, yielding comparable specific growth rates of (3.66 ± 0.28) × 10 −2 h -1 (30 mL), (3.27 ± 0.40) × 10 −2 h -1 (1 L), and 3.50 × 10 −2 h -1 (5 L), and reaching a total yield of 4.23 × 10 9 cells in the 5-L bioreactor (a single run). These findings demonstrate that fluffy nanofiber scaffolds enable a scale-up strategy that reproduces the cellular microenvironment in a manner that is less affected by scale-dependent physical forces. This concept provides a new framework for designing scalable culture environments applicable not only to cell therapy manufacturing but also to culture supernatant production and cell–based food materials.


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:
Osteoarthritis (OA) remains without disease‐modifying sssstherapies, in part due to biological heterogeneity and a hostile joint microenvironment that undermines one‐size‐fits‐all interventions. Extracellular vesicles (EVs) play a dual role in OA pathophysiology: endogenous EVs disseminate pro‐inflammatory and catabolic signals that propagate cartilage degeneration, whereas therapeutic EVs most commonly derived from regenerative cell sources can deliver anti‐inflammatory and anabolic cues. We frame this contrast as the EV paradox and argue that it represents a central translational challenge explaining why robust preclinical efficacy has not yet translated into consistent clinical benefit. We synthesize current evidence on EV biology in joint tissues, outcomes across preclinical models, and early human studies that demonstrate safety but limited efficacy. This analysis highlights key barriers to translation, including impaired EV function within inflamed and mechanically active joints, rapid clearance and limited tissue targeting, mismatch between animal models and human disease, and insufficient standardization of EV potency. Building on these insights, we propose a precision‐medicine roadmap that emphasizes patient stratification, rational EV design, improved delivery strategies, and manufacturing frameworks linked to mechanism‐anchored endpoints. Together, this framework reframes the EV paradox from a translational obstacle into a design principle for developing disease‐modifying EV‐based therapies for OA.


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:
Succinic acid has been considered an important molecule in the transition of chemical manufacturing from fossil‐based to sustainable and future‐proof processes. While there has been extensive research on biotechnological succinic acid production from biomass, attempts to roll out bio‐succinic acid are impeded by its high price and remaining sustainability issues. Both drawbacks are interconnected and can be traced back to the used feedstocks and a wasteful expenditure of acid and base, among others. In this opinion, we discuss biochemical principles and metabolic pathways of next‐generation carbon assimilation and low‐pH fermentations to address these drawbacks. For this reason, we chart the potential for producing succinic acid from sustainable next‐generation feedstocks based on electron, carbon and ATP balances as well as relevant thermodynamic considerations. Furthermore, we summarize key advances in low‐pH succinic acid synthesis using acid‐tolerant yeasts and assess the suitability of selected acid tolerance mechanisms for industrial application. Eventually, we aim to inspire researchers to synthesize innovative approaches to realize competitive and sustainable biotechnological succinic acid production. Keywords: industrial biotechnology, low‐pH fermentation, metabolic engineering, one carbon metabolism, succinic acid

Candidates:
A. In this opinion, we discuss biochemical principles and metabolic pathways of next‐generation carbon assimilation and low‐pH fermentations to address these drawbacks.
B. While there has been extensive research on biotechnological succinic acid production from biomass, attempts to roll out bio‐succinic acid are impeded by its high price and remaining sustainability issues.
C. The evidence does not state that succinic acid has been considered an important molecule in the transition of chemical manufacturing from fossil‐based to sustainable and future‐proof processes.
D. The evidence does not state that in this opinion, we discuss biochemical principles and metabolic pathways of next‐generation carbon assimilation and low‐pH fermentations to address these drawbacks.
E. Succinic acid has been considered an important molecule in the transition of chemical manufacturing from fossil‐based to sustainable and future‐proof processes.
F. While there has been extensive research on biotechnological succinic acid production from biomass, attempts to roll out bio‐succinic acid are not impeded by its high price and remaining sustainability issues.
G. Both drawbacks are interconnected and can be traced back to the used feedstocks and a wasteful expenditure of acid and base, among others.
H. Both drawbacks are not interconnected and can be traced back to the used feedstocks and a wasteful expenditure of acid and base, among 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:
Monoclonal antibodies (mAbs) constitute the most rapidly expanding and therapeutically impactful class of biological medicines, driven by their exceptional target specificity, modular engineering capabilities, predictable pharmacokinetics facilitated by neonatal Fc receptor (FcRn) recycling, and favorable safety profiles attributable to mechanism-based pharmacology. As foundational patents on blockbuster antibody therapeutics expire, biosimilar antibodies have become a vital mechanism to enhance global accessibility, improve healthcare sustainability, and promote market competition. Simultaneously, regulatory agencies worldwide are substantially restructuring development expectations to emphasize analytical precision, mechanistic understanding, human-relevant methodologies, and ethical nonclinical practices aligned with the 3Rs principles of replacement, reduction, and refinement. This comprehensive review analyzes the convergence of innovator and biosimilar antibody development toward an analytics-first comparability paradigm, mechanism-based safety assessment, and targeted clinical confirmation based on residual uncertainty rather than prescriptive requirements. It systematically incorporates regulatory positions from the United States Food and Drug Administration (FDA), European Medicines Agency (EMA), World Health Organization (WHO), Japan’s Pharmaceuticals and Medical Devices Agency (PMDA), Health Canada, and the International Council for Harmonisation (ICH), together with contemporary peer-reviewed research literature. Focus is given to the FDA Modernization Act 2.0, which abolished statutory mandates for animal testing, and recent FDA draft guidance—which are provisional and open to revision pending finalization—addressing streamlined nonclinical safety studies for monospecific antibodies and science-based approaches to waiving comparative clinical efficacy studies (CES). The scientific boundaries distinguishing products amenable to streamlined development from those requiring tailored approaches are delineated, including the exclusion of polyclonal antibody preparations from biosimilar frameworks and the analytical complexities associated with conjugated antibodies and multispecific constructs. Important limitations of analytics-first approaches are also addressed, including scenarios involving heightened immunogenicity risk, novel targets with limited clinical experience, or insufficiently validated pharmacodynamic markers. These developments are examined alongside counterarguments and residual areas of regulatory skepticism. While the trajectory of regulatory reform appears to favor evidence-efficient, science-driven models for antibody development, this review explicitly distinguishes between established regulatory consensus, emerging draft guidance positions, and the author’s scholarly interpretation, recognizing that the pace and scope of regulatory change remain subject to ongoing deliberation.

Candidates:
A. The evidence does not state that as foundational patents on blockbuster antibody therapeutics expire, biosimilar antibodies have become a vital mechanism to enhance global accessibility, improve healthcare sustainability, and promote market competition.
B. The evidence does not state that monoclonal antibodies (mAbs) constitute the most rapidly expanding and therapeutically impactful class of biological medicines, driven by their exceptional target specificity, modular engineering capabilities, predictable pharmacokinetics facilitated by neonatal Fc receptor (FcRn) recycling, and favorable safety profiles attributable to mechanism-based pharmacology.
C. Monoclonal antibodies (mAbs) constitute the most rapidly expanding and therapeutically impactful class of biological medicines, driven by their exceptional target specificity, modular engineering capabilities, predictable pharmacokinetics facilitated by neonatal Fc receptor (FcRn) recycling, and favorable safety profiles attributable to mechanism-based pharmacology.
D. As foundational patents on blockbuster antibody therapeutics expire, biosimilar antibodies have become a vital mechanism to enhance global accessibility, improve healthcare sustainability, and promote market competition.
E. Simultaneously, regulatory agencies worldwide are substantially restructuring development expectations to emphasize analytical precision, mechanistic understanding, human-relevant methodologies, and ethical nonclinical practices aligned with the 3Rs principles of replacement, reduction, and refinement.
F. Simultaneously, regulatory agencies worldwide are substantially restructuring development expectations to emphasize analytical precision, mechanistic understanding, human-relevant methodologies, and ethical nonclinical practices aligned with the 4Rs principles of replacement, reduction, and refinement.
G. The evidence does not state that this comprehensive review analyzes the convergence of innovator and biosimilar antibody development toward an analytics-first comparability paradigm, mechanism-based safety assessment, and targeted clinical confirmation based on residual uncertainty rather than prescriptive requirements.
H. This comprehensive review analyzes the convergence of innovator and biosimilar antibody development toward an analytics-first comparability paradigm, mechanism-based safety assessment, and targeted clinical confirmation based on residual uncertainty rather than prescriptive requirements.


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 spectral preprocessing step of data derived from Raman spectroscopy is important for chemometric models’ calibration, which allows real-time monitoring of pharmaceutical bioprocesses. Thus, the present work aimed to establish, with statistical criteria, the best combination of spectral filters for the biochemical monitoring of the baculovirus/ Sf9 insect cell system. The production of rabies virus-like particles was used as a model. Combining moving window smoothing and offset baseline correction spectral filters demonstrated the highest efficacy in biochemically monitoring the baculovirus/ Sf9 insect cell system using Raman spectroscopic data and Partial Least Squares regression. However, when applying Artificial Neural Network modeling, a different set of filters proved more effective: wavelet denoise spectral smoothing, asymmetric least square baseline correction, standard normal variate normalization, and quadratic first derivative. The simulation results demonstrate that by following these guidelines for spectral preprocessing, cell viability, glucose, lactate, glutamine, glutamate, and ammonium can be satisfactorily monitored in real-time, but not the density of viable cells. The absolute errors for cell viability, glucose, lactate, glutamine, glutamate, and ammonium were lower than 12%, 0.47 g L − 1 , 8.95 mg L − 1 , 0.11 g L − 1 , 0.10 g L − 1 , 3.21 mg L − 1 , respectively, which are suitable for bioprocess in-line monitoring and control through a soft sensor. The online version contains supplementary material available at 10.1007/s00449-026-03301-1. Keywords: Artificial neural network, Bioprocess monitoring, Partial least squares, Raman spectroscopy, SARS-CoV-2, Virus-like particles

Candidates:
A. Combining moving window smoothing and offset baseline correction spectral filters demonstrated the highest efficacy in biochemically monitoring the baculovirus/ Sf9 insect cell system using Raman spectroscopic data and Partial Least Squares regression.
B. The production of rabies virus-like particles was not used as a model.
C. The production of rabies virus-like particles was used as a model.
D. Combining moving window smoothing and offset baseline correction spectral filters demonstrated the highest efficacy in biochemically monitoring the baculovirus/ Sf10 insect cell system using Raman spectroscopic data and Partial Least Squares regression.
E. Thus, the present work aimed to establish, with statistical criteria, the best combination of spectral filters for the biochemical monitoring of the baculovirus/ Sf10 insect cell system.
F. The spectral preprocessing step of data derived from Raman spectroscopy is important for chemometric models’ calibration, which allows real-time monitoring of pharmaceutical bioprocesses.
G. The spectral preprocessing step of data derived from Raman spectroscopy is not important for chemometric models’ calibration, which allows real-time monitoring of pharmaceutical bioprocesses.
H. Thus, the present work aimed to establish, with statistical criteria, the best combination of spectral filters for the biochemical monitoring of the baculovirus/ Sf9 insect cell system.


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 fermentation of industrial wastes derived from water recovery (pretreated microalgae biomass, PTMB), biodiesel (glycerol, GLY), and ethanol (vinasse, VIN)) is a promising sustainable alternative for producing volatile fatty acids (VFA). In this study, controlled fermentation experiments were performed using PTMB-VIN and PTMB-GLY mixtures with varying substrate concentrations and retention times to optimize the VFA yield and production efficiency. Acid fermentation was optimized at a 3-day hydraulic retention time, and methanogenic reactions were enhanced at longer hydraulic retention times. Increasing the organic loading rate resulted in a high VFA yield (13.10 g VFA-COD . L −1 ) and a conversion rate of approximately 40%. GLY fermentation followed the oxidative and reductive pathways at a balanced redox potential. PTMB is linked to the oxidative pathway, and GLY is involved in the reductive pathway. Tests with PTMB-GLY resulted in limiting conditions, but significant observations regarding the metabolic pathway and the effect of PTMB were made. Keywords: Dark fermentation; Acidogenesis; Cosubstrate; Hydraulic retention time; 1,3 propanediol; Metabolic pathways

Candidates:
A. Increasing the organic loading rate resulted in a high VFA yield (13.10 g VFA-COD .
B. In this study, controlled fermentation experiments were not performed using PTMB-VIN and PTMB-GLY mixtures with varying substrate concentrations and retention times to optimize the VFA yield and production efficiency.
C. Acid fermentation was optimized at a 4-day hydraulic retention time, and methanogenic reactions were enhanced at longer hydraulic retention times.
D. Acid fermentation was optimized at a 3-day hydraulic retention time, and methanogenic reactions were enhanced at longer hydraulic retention times.
E. The fermentation of industrial wastes derived from water recovery (pretreated microalgae biomass, PTMB), biodiesel (glycerol, GLY), and ethanol (vinasse, VIN)) is a promising sustainable alternative for producing volatile fatty acids (VFA).
F. Increasing the organic loading rate resulted in a high VFA yield (14.10 g VFA-COD .
G. In this study, controlled fermentation experiments were performed using PTMB-VIN and PTMB-GLY mixtures with varying substrate concentrations and retention times to optimize the VFA yield and production efficiency.
H. The fermentation of industrial wastes derived from water recovery (pretreated microalgae biomass, PTMB), biodiesel (glycerol, GLY), and ethanol (vinasse, VIN)) is not a promising sustainable alternative for producing volatile fatty acids (VFA).


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: ["97%", "50%", "51%", "98%"]

Evidence:
Chinese hamster ovary (CHO) cells constitute the industry-standard platform for the production of complex therapeutic proteins, yet genomic heterogeneity arising from random integration leads to clonal variability and unstable expression, necessitating a robust cell line development process to efficiently isolate stable, high-expressing clones. By employing an optimized integration strategy, recombinant cell line performance can be enhanced through improved genomic stability and increased productivity. In this study, GFP reporter analysis demonstrated that the PiggyBac system significantly boosts the yield of both stable and high-expression clones. Subsequently, transposase modified with a nuclear localization signal (NLS) exhibited superior stability in recombinant cell lines and polyclonal pools. The nucleoplasmin NLS resulted in transgene integration into genomic loci that promote enhanced and more stable expression, thereby improving clonal distribution. Furthermore, this strategy also increased recombinant mAb expression by 97% in pools while enhancing average and specific productivity in derived cell lines. Additionally, recombinant suspension cells generated with the optimized system exhibited comparable performance, with over 50% of minipools showing robust growth. Overall, these findings highlight the promising utility of the NLS-optimized PiggyBac system for improving transgene stability and expression while streamlining the cell line screening process. The online version contains supplementary material available at 10.1186/s40643-026-01056-x. Keywords: Stability, Cell line construction, Transposon, PiggyBac system, Nuclear localization signal, Production


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:
Combinatorial barcoding technologies for single-cell nucleotide sequencing, such as split-pool ligation protocols, involve sequential rounds of cell barcoding to uniquely tag individual cells. The rapid adoption of combinatorial barcoding in recent years is due in part to its scalability across cells and samples. However, small shifts in barcode positions within sequencing reads caused by technical artifacts, e.g. during barcode incorporation or synthesis, can impact the accurate assignment of reads to cell barcodes. Existing processing tools typically assume barcodes contain fixed-length nucleotide sequences located at fixed positions within reads, overlooking any positional variability. Consequently, reads containing truncated or mispositioned barcodes are discarded during initial data processing steps leading to significant data loss. To solve this limitation and maximize the retention of sequencing reads from single-cell combinatorial barcoding experiments, we introduce scarecrow . This tool screens a subsample of reads to generate position-specific barcode profiles, which are then used to flexibly identify barcode sequences in each read whilst accounting for positional errors, a phenomenon we refer to as “jitter”. Barcode matches are then prioritized to minimize nucleotide mismatches and the degree of jitter. These initial profiles are subsequently used to extract and error correct barcode combinations in high throughput sequencing libraries. By incorporating jitter into barcode error correction, scarecrow enables greater data recovery and improved downstream single-cell analyses. Scarecrow is fully open access, implemented in Python, and generates output files using standardized sequence file formats for maximal interoperability. A detailed explanation of the scarecrow workflow can be found in the supplementary materials. Scarecrow is freely available on GitHub https://github.com/MorganResearchLab/scarecrow and Zenodo https://doi.org/10.5281/zenodo.18621784 .

Candidates:
A. The rapid adoption of combinatorial barcoding in recent years is due in part to its scalability across cells and samples.
B. However, small shifts in barcode positions within sequencing reads caused by technical artifacts, e.g.
C. The evidence does not state that combinatorial barcoding technologies for single-cell nucleotide sequencing, such as split-pool ligation protocols, involve sequential rounds of cell barcoding to uniquely tag individual cells.
D. Combinatorial barcoding technologies for single-cell nucleotide sequencing, such as split-pool ligation protocols, involve sequential rounds of cell barcoding to uniquely tag individual cells.
E. during barcode incorporation or synthesis, can impact the accurate assignment of reads to cell barcodes.
F. The rapid adoption of combinatorial barcoding in recent years is not due in part to its scalability across cells and samples.
G. during barcode incorporation or synthesis, cannot impact the accurate assignment of reads to cell barcodes.
H. The evidence does not state that however, small shifts in barcode positions within sequencing reads caused by technical artifacts, e.g.


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:
Mammalian cell cultures are widely used for producing complex biopharmaceuticals that require human-like post-translational modifications, such as antibody-based therapeutics. Traditionally, serum-supplemented media support high cell viability and productivity; however, regulatory and scientific requirements demand serum-free conditions for clinical-grade manufacture. Recently, a novel fusion protein, the anti-huCD20(hγ1)-IL2no-alpha immunocytokine (IC), was presented as a promising therapeutic alternative, mostly for relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL) patients, considering currently approved therapies. Three Chinese hamster ovary clones (K1 strain) producing the anti-huCD20(hγ1)-IL2no-alpha IC were generated and adapted to serum-free suspension culture. We performed a kinetic characterization of one clone in two culture media with different nutritional compositions, evaluating cell growth, productivity, cell cycle progression and mTOR signaling. The IC was purified by Protein A, then evaluated for identity, aggregation profile, CD20 recognition, CTLL-2 cytokine activity, ex vivo B-cell depletion in PBMC from r/r B-NHL patients and antitumor efficacy in immunocompetent C57BL/6 mice bearing EL4-hCD20- cells. The results demonstrated noticeable differences in cell growth and productivity in both batch and pseudo-perfusion performance, likely due to an influence on cell-cycle progression and mTOR signaling. The purified IC maintained its structural integrity while exhibiting an improved aggregation profile compared to serum-containing cultures. Furthermore, key biological activities, including B-cell depletion and antitumoral effects, remained intact. This research highlights the successful serum-free production of a functional anti-huCD20(hγ1)-IL2no-alpha IC, reinforcing its potential for biopharmaceutical development.


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: ["3.7 g/L", "2.7 g/L", "44%", "4.3 g/L", "10.2 g/L", "11.2 g/L", "5.3 g/L", "45%"]

Evidence:
Soybean whey wastewater (SWW), a rich source of soybean whey protein (SWP), is prone to microbial rancidity, posing environmental and resource challenges. This study explores the causes of rancidity—characterized by a pungent, sour, and putrid odor—in the effluents of sealed buffer tank during SWP recovery via pneumatic flotation. Metagenome, bacterial diversity, and HPLC analyses showed the obligate anaerobe Megasphaera spp. dominated rancid effluents (up to 44% abundance), consumed lactate (decreasing from 10.2 g/L in influent to 2.7 g/L in effluent), and produced malodorous propionate and butyrate (up to 3.6 and 4.3 g/L, respectively). Three mitigation strategies were assessed: (1) full‐scale high‐throughput aeration—likely effective but energy‐ and cost‐intensive; (2) local aeration—low‐cost but weakly inhibitory; and (3) microbial intervention using the probiotic Enterococcus faecium LBSW, which colonizes the buffer tank, with localized aeration used only if microbial control fails. Strategy (3) was adopted for its energy and cost efficiency, successfully reducing pollution and supporting SWP recovery. Although the biosafety of E. faecium LBSW in food applications requires caution, the recovered SWP is primarily intended for animal feed, and subsequent high‐temperature drying and sterilization (> 120°C) also offer potential for food‐grade 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:
Chimeric antigen receptor (CAR) T cell therapies have achieved clinical success in autologous treatment of hematological malignancies. However, their broader application remains limited. Beyond the biological challenges associated with the immunosuppressive microenvironment of solid tumors and the graft-versus-host disease risks inherent to allogeneic settings, the widespread adoption of CAR T cells is hindered by the high costs, long and complex vein-to-vein timelines and variability in product quality. Rapid CAR T cell manufacturing has emerged as an alternative paradigm that prioritizes shortened production workflows and preservation of naïve and stem-like T cell phenotypes with superior in vivo expansion, persistence and anti-tumor efficacy. This review examines rapid CAR T cell manufacturing from a bioprocess engineering perspective, focusing on how critical process parameters can be explored to shape CAR T cell phenotype and function within shortened timelines. We highlight key technological enablers, including automation, microfluidic systems, process analytical technologies, artificial intelligence-driven bioprocess control, quality control methodologies as well as safety considerations unique to accelerated workflows. Emerging CAR T cell manufacturing models, such as point-of-care production and in vivo CAR T cells generation, are also discussed. These insights outline engineering strategies to enable faster, more consistent and clinically effective CAR T cells. Keywords: CAR T cell, Stem-like CAR T cells, Rapid manufacturing, Bioprocess parameters, Point‑of‑care manufacturing, Bioprocess control

Candidates:
A. The evidence does not state that chimeric antigen receptor (CAR) T cell therapies have achieved clinical success in autologous treatment of hematological malignancies.
B. The evidence does not state that however, their broader application remains limited.
C. Rapid CAR T cell manufacturing has emerged as an alternative paradigm that prioritizes shortened production workflows and preservation of naïve and stem-like T cell phenotypes with superior in vivo expansion, persistence and anti-tumor efficacy.
D. Chimeric antigen receptor (CAR) T cell therapies have achieved clinical success in autologous treatment of hematological malignancies.
E. However, their broader application remains limited.
F. Beyond the biological challenges associated with the immunosuppressive microenvironment of solid tumors and the graft-versus-host disease risks inherent to allogeneic settings, the widespread adoption of CAR T cells is hindered by the high costs, long and complex vein-to-vein timelines and variability in product quality.
G. Beyond the biological challenges associated with the immunosuppressive microenvironment of solid tumors and the graft-versus-host disease risks inherent to allogeneic settings, the widespread adoption of CAR T cells is not hindered by the high costs, long and complex vein-to-vein timelines and variability in product quality.
H. The evidence does not state that rapid CAR T cell manufacturing has emerged as an alternative paradigm that prioritizes shortened production workflows and preservation of naïve and stem-like T cell phenotypes with superior in vivo expansion, persistence and anti-tumor efficacy.


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: ["64%", "41–50%", "63%", "40–50%"]

Evidence:
Chitinases from Trichoderma species exhibit strong antifungal activity and high biocontrol potential, yet their industrial utilization has been constrained by low heterologous secretion efficiency and costly purification processes. In this study, the Generally Recognized As Safe yeast Saccharomyces cerevisiae Y2805 was engineered to secrete chitinase Tch36 from a Korean isolate of T. atroviride , using a rice α-amylase signal peptide under a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. When cultivated in glycerol–colloidal chitin medium, the recombinant yeast exhibited approximately a fivefold increase in measurable chitinase activity relative to the SC mock control, whereas the empty-vector control showed only a modest increase. The crude, non-concentrated culture filtrate (approximately 1000 U L⁻ 1 ) displayed statistically significant antifungal activity against 12 fungal species, including 9 phytopathogens and 3 opportunistic Aspergillus species. Among the phytopathogens, Fusarium graminearum was one of the most strongly inhibited species, showing approximately 40–50% suppression of colonial growth on solid medium. In liquid culture, microscopy-based germination assays revealed ≥ 63% inhibition of early hyphal elongation in Botrytis cinerea and A. niger . All antifungal effects were evaluated relative to filtrates from the empty-vector control prepared under equivalent dilution conditions. The culture filtrate also enhanced protoplast formation, providing direct evidence of chitinase-associated cell wall weakening. Collectively, these results establish a purification-free, yeast-based heterologous secretion platform capable of producing active Trichoderma chitinase with inhibitory effects on diverse plant- and animal-associated fungi. This strategy has practical potential for biocontrol applications and for sustainable bioprocessing technologies based on microbial chitinase production. The online version contains supplementary material available at 10.1186/s40643-026-01054-z. Keywords: Saccharomyces cerevisiae , Trichoderma atroviride , Chitinase, Yeast secretion, Antifungal biocontrol, Extracellular enzyme production


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:
Optimization of biotechnological processes is traditionally limited by time-consuming trial-and-error approaches and the complexity of simultaneously optimizing multiple, often conflicting objectives. This applies particularly to plant tissue culture medium design, which therefore serves as the application case in this study. Recent advances in machine learning and evolutionary algorithms offer powerful alternatives, yet 80% of published studies rely on licensed software, and systematic data-driven optimization frameworks remain scarce. This creates significant barriers to adoption in both academic and commercial plant biotechnology. We introduce ADAM (Advanced Design and AI-Driven Modeling for Plant Tissue Culture Media Optimization), an open-access, web-based platform that transforms protocol development into a data-driven computational process. ADAM implements a complete ML-EA workflow through five integrated modules: 1. Design of Experiments (five different concepts) for systematic parameter exploration, 2. Data Preparation with automated quality control, and 3. Model Building using nine machine learning algorithms with automated selection. The platform enables Optimization (4.) through four advanced evolutionary algorithms (genetic algorithm, particle swarm optimization, NSGA-II, SMS-EMOA) for single- and multi-objective problems, with Evaluation (5.) tools to compare original versus optimized solutions. Validation across two plant tissue culture applications showed that ADAM’s models matched or exceeded the predictive performance of manually optimized approaches in the original studies. The platform successfully identified multiple optimal culture conditions balancing conflicting objectives, providing experimentally testable predictions that reduce the trial-and-error cycle. Deployed as a browser-based application requiring neither specialized hardware nor software licenses, ADAM democratizes advanced AI optimization for plant biotechnology, eliminating traditional barriers to entry while maintaining the rigor and flexibility required for scientific research. The online version contains supplementary material available at 10.1186/s13007-026-01534-5. Keywords: Plant tissue culture, Machine learning, Predictive modelling, Evolutionary algorithms, Culture media optimization

Candidates:
A. Recent advances in machine learning and evolutionary algorithms offer powerful alternatives, yet 80% of published studies rely on licensed software, and systematic data-driven optimization frameworks remain scarce.
B. This creates significant barriers to adoption in both academic and commercial plant biotechnology.
C. Optimization of biotechnological processes is traditionally limited by time-consuming trial-and-error approaches and the complexity of simultaneously optimizing multiple, often conflicting objectives.
D. The evidence does not state that this applies particularly to plant tissue culture medium design, which therefore serves as the application case in this study.
E. The evidence does not state that this creates significant barriers to adoption in both academic and commercial plant biotechnology.
F. Recent advances in machine learning and evolutionary algorithms offer powerful alternatives, yet 81% of published studies rely on licensed software, and systematic data-driven optimization frameworks remain scarce.
G. This applies particularly to plant tissue culture medium design, which therefore serves as the application case in this study.
H. Optimization of biotechnological processes is not traditionally limited by time-consuming trial-and-error approaches and the complexity of simultaneously optimizing multiple, often conflicting objectives.


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:
Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable efficient conversion of volatile or poorly water-soluble substrates. Acinetobacter sp. Tol 5 is a highly adhesive bacterium capable of utilizing various hydrocarbons, making it a promising chassis candidate for gas-phase bioprocesses. However, Gas-phase bioprocesses expose cells to fluctuating humidity and transient desiccation that can compromise viability and catalytic performance. In Gram-negative bacteria, especially in pathogens, desiccation is known to impose multifactorial stress, such as loss of cellular water and energetics, damage to DNA and proteins, oxidative stress, and disruption of the cell envelope. In contrast, for chassis strains used in or being considered for gas-phase bioprocesses, their desiccation tolerance including robustness of biocatalytic activity following humidity fluctuations and responses to desiccation stress remain incompletely defined. Here, we evaluated the viability, energy status, and gas-phase toluene degradation of Tol 5 as a chassis for gas-phase processes after controlled desiccation (8%, 52%, and > 95% RH), in comparison with Acinetobacter baylyi ADP1, Pseudomonas putida , and Escherichia coli . To minimize adhesion-related bias in post-desiccation measurements, we used an ataA -deficient Tol 5 mutant (Tol 5 Δ ataA ) for the assays. Acinetobacter strains maintained high viability during desiccation for 16 days, whereas P. putida and E. coli showed significant loss of viability at 8% RH and 52% RH. Intracellular ATP measurements further indicated that desiccation reduced intracellular ATP in all strains, but E. coli rapidly exhausted ATP at 8% and 52% RH, whereas Acinetobacter strains and P. putida retained intracellular ATP. In a gas-phase toluene degradation assay, immobilized Tol 5 retained higher toluene-degrading activity after desiccation than P. putida mt-2. Transcriptome profiling revealed a multilayered Tol 5 response involving DNA and RNA maintenance, cell envelope trafficking and remodeling, redox-responsive functions, and broad repression of growth-associated metabolism. Our results highlight Acinetobacter , particularly Tol 5, as a promising chassis candidate for gas-phase bioprocesses and suggest potential mechanistic targets for stabilizing bacterial cells under low-water-activity conditions. The online version contains supplementary material available at 10.1186/s13036-026-00668-3.

Candidates:
A. In Gram-negative bacteria, especially in pathogens, desiccation is known to impose multifactorial stress, such as loss of cellular water and energetics, damage to DNA and proteins, oxidative stress, and disruption of the cell envelope.
B. Tol 6 is a highly adhesive bacterium capable of utilizing various hydrocarbons, making it a promising chassis candidate for gas-phase bioprocesses.
C. However, Gas-phase bioprocesses expose cells to fluctuating humidity and transient desiccation that can compromise viability and catalytic performance.
D. Tol 5 is a highly adhesive bacterium capable of utilizing various hydrocarbons, making it a promising chassis candidate for gas-phase bioprocesses.
E. However, Gas-phase bioprocesses expose cells to fluctuating humidity and transient desiccation that cannot compromise viability and catalytic performance.
F. In Gram-negative bacteria, especially in pathogens, desiccation is not known to impose multifactorial stress, such as loss of cellular water and energetics, damage to DNA and proteins, oxidative stress, and disruption of the cell envelope.
G. Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable efficient conversion of volatile or poorly water-soluble substrates.
H. The evidence does not state that gas-phase bioprocesses that immobilize microbial cells on solid carriers enable efficient conversion of volatile or poorly water-soluble substrates.


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: ["1001 mL", "1000 mL", "10 mL", "30 °C", "11 mL", "35 °C", "36 °C", "31 °C"]

Evidence:
The increasing reliance on fossil fuels for global energy production has intensified greenhouse gas emissions, highlighting the need for sustainable energy alternatives. Hydrogen is considered a promising green fuel due to its high energy density and conversion efficiency. Among various production pathways, microalgae-based biohydrogen generation via biophotolysis is particularly attractive owing to its high biomass productivity, adaptability to diverse water sources, flue gas mitigation potential, and low land requirements.This study investigates the effects of key microalgal growth parameters on biohydrogen production by Chlorella sp. through biophotolysis. The impacts of nitrogen purging during the transition from aerobic to anaerobic conditions, different photoperiod regimes (continuous illumination, continuous darkness, and a light–dark cycle), glucose supplementation (5, 10, and 15 g L⁻ 1 ), and temperature (25, 30, and 35 °C) were systematically evaluated. Microalgal cell density was monitored during hydrogen production to elucidate its relationship with hydrogen yield. Initial experiments were conducted in 10 mL test tubes to identify optimal conditions, which were subsequently applied to scale-up experiments in a 1000 mL jacketed reactor. Nitrogen purging significantly enhanced hydrogen production by removing oxygen and activating hydrogenase, resulting in a peak hydrogen concentration of 11 ppm. Continuous illumination yielded higher hydrogen levels than darkness and light–dark cycling. Glucose addition substantially increased hydrogen production, with the highest yield observed at 15 g L⁻ 1 (30 ppm). An optimal temperature of 30 °C also maximized hydrogen production. Under these conditions, hydrogen production increased as cell density decreased due to metabolic shifts. Scale-up experiments achieved a 405-fold increase in hydrogen yield, demonstrating the scalability potential of the process. These findings emphasize the importance of optimizing algal growth conditions to balance microalgal growth and biohydrogen production for future industrial applications. Keywords: Biohydrogen, Microalgae, Biophotolysis, Cell growth, Nitrogen purging, Photoperiod, Carbon source


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 biomass of Quillaja saponaria is a well-characterized source of triterpenic saponins, a family of amphiphilic glycosides with broad industrial and biomedical relevance. Among these, the highly purified fraction QS-21, and the related fractions QHA and QHC, have strong immunostimulant properties and have become key adjuvant components of several FDA-approved human vaccines. The expanding global use of QS-21-based adjuvants has raised concerns regarding long-term availability and sustainability, as commercial production currently relies exclusively on extraction from Q. saponaria biomass sourced in Chile. This review summarizes the historical development, chemical features, and immunological relevance of QS-21, and critically evaluates existing and emerging production technologies in terms of a projected production benchmark of 50 Kg per year (corresponding to at least one billion 50 µg doses). We conclude that appropriate management of wild Quillaja forests can sustain kilogram-scale QS-21 purification, but to meet increasing demand, clonal plantation forestry will be the most practical and scalable strategy in the near future. Alternative approaches, including plant cell culture, engineered microorganisms, and chemical synthesis, remain scientifically promising but currently face technical and economic limitations for large-scale pharmaceutical production.

Candidates:
A. Among these, the highly purified fraction QS-22, and the related fractions QHA and QHC, have strong immunostimulant properties and have become key adjuvant components of several FDA-approved human vaccines.
B. The expanding global use of QS-22-based adjuvants has raised concerns regarding long-term availability and sustainability, as commercial production currently relies exclusively on extraction from Q.
C. The biomass of Quillaja saponaria is a well-characterized source of triterpenic saponins, a family of amphiphilic glycosides with broad industrial and biomedical relevance.
D. The expanding global use of QS-21-based adjuvants has raised concerns regarding long-term availability and sustainability, as commercial production currently relies exclusively on extraction from Q.
E. Among these, the highly purified fraction QS-21, and the related fractions QHA and QHC, have strong immunostimulant properties and have become key adjuvant components of several FDA-approved human vaccines.
F. The biomass of Quillaja saponaria is not a well-characterized source of triterpenic saponins, a family of amphiphilic glycosides with broad industrial and biomedical relevance.
G. This review summarizes the historical development, chemical features, and immunological relevance of QS-22, and critically evaluates existing and emerging production technologies in terms of a projected production benchmark of 50 Kg per year (corresponding to at least one billion 50 µg doses).
H. This review summarizes the historical development, chemical features, and immunological relevance of QS-21, and critically evaluates existing and emerging production technologies in terms of a projected production benchmark of 50 Kg per year (corresponding to at least one billion 50 µg doses).


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 systematic exploration of novel bioactive compounds with superior functional properties is critical for driving innovations in agriculture, healthcare, and related fields, thereby becoming essential for advancing sustainable biotechnological solutions. Nonprotein amino acids (NPAAs), functional amino acids not incorporated into proteins, exhibit unique physiological activities and provide distinctive advantages in nutritional enhancement, functional product formulation, and food/feed processing. These attributes challenge the conventional perception of proteins as mere nutritional carriers, positioning NPAAs as promising bioproducts for biosynthesis and functional applications in agriculture, food, and medicine. This review summarizes the classification of the available NPAAs based on their synthetic substrates for the first time and then outlines their diverse functional roles. A comprehensive analysis of recent advances in biosynthetic pathways, engineering strategies, and production level demonstrates their primary research progress in the laboratory phase. The further sustainable biomanufacturing of NPAAs is hampered by several challenges, including poorly elucidated biosynthetic mechanisms, limited robustness and low productivity of microbial strains, and difficulties in scaling up production for industrial applications. Addressing these bottlenecks will require innovative strategies and technologies to facilitate the translation of NPAA production from bench to industry. This review offers valuable insights into the potential of NPAAs in the development of next-generation bioproducts of nutrition, immune regulation, antioxidant defense, and intestinal homeostasis maintenance, suggesting a promising direction for microbial production of high-performance bioactive molecules in agricultural synthetic biomanufacturing.

Candidates:
A. The evidence does not state that these attributes challenge the conventional perception of proteins as mere nutritional carriers, positioning NPAAs as promising bioproducts for biosynthesis and functional applications in agriculture, food, and medicine.
B. Nonprotein amino acids (NPAAs), functional amino acids not incorporated into proteins, exhibit unique physiological activities and provide distinctive advantages in nutritional enhancement, functional product formulation, and food/feed processing.
C. The systematic exploration of novel bioactive compounds with superior functional properties is not critical for driving innovations in agriculture, healthcare, and related fields, thereby becoming essential for advancing sustainable biotechnological solutions.
D. The evidence does not state that this review summarizes the classification of the available NPAAs based on their synthetic substrates for the first time and then outlines their diverse functional roles.
E. These attributes challenge the conventional perception of proteins as mere nutritional carriers, positioning NPAAs as promising bioproducts for biosynthesis and functional applications in agriculture, food, and medicine.
F. This review summarizes the classification of the available NPAAs based on their synthetic substrates for the first time and then outlines their diverse functional roles.
G. The evidence does not state that nonprotein amino acids (NPAAs), functional amino acids not incorporated into proteins, exhibit unique physiological activities and provide distinctive advantages in nutritional enhancement, functional product formulation, and food/feed processing.
H. The systematic exploration of novel bioactive compounds with superior functional properties is critical for driving innovations in agriculture, healthcare, and related fields, thereby becoming essential for advancing sustainable biotechnological solutions.


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:
Pre-use post-sterilization integrity testing (PUPSIT) has emerged as a critical standard in biopharmaceutical manufacturing, driven by the revised EU GMP Annex 1 and global regulatory harmonization. PUPSIT aims to verify the integrity of sterilized filters before use, reducing the risks of defect masking and safeguarding drugs against contamination. However, implementing PUPSIT introduces significant technical and operational challenges, including more complex wetting and venting procedures that may bring increased risk of contamination and susceptibility to human error. This review outlines regulatory requirements and risk-based rationales for the PUPSIT evaluation process and discusses the integration of PUPSIT within a broader contamination control strategy. Design strategies for single-use systems emphasize simplicity, error-proofing, and effective wetting and flushing methods. A comparative analysis of single-use PUPSIT systems including manual, shadowboard-assisted, and automated PUPSIT systems illustrates how process flexibility, consistency, and safety can be achieved. Ultimately, successful PUPSIT implementation requires a balance of compliance, operational efficiency, and environmental stewardship, supported by documented risk assessments and validated process controls.

Candidates:
A. Pre-use post-sterilization integrity testing (PUPSIT) has emerged as a critical standard in biopharmaceutical manufacturing, driven by the revised EU GMP Annex 1 and global regulatory harmonization.
B. The evidence does not state that this review outlines regulatory requirements and risk-based rationales for the PUPSIT evaluation process and discusses the integration of PUPSIT within a broader contamination control strategy.
C. PUPSIT aims to verify the integrity of sterilized filters before use, reducing the risks of defect masking and safeguarding drugs against contamination.
D. However, implementing PUPSIT introduces significant technical and operational challenges, including more complex wetting and venting procedures that may bring increased risk of contamination and susceptibility to human error.
E. The evidence does not state that however, implementing PUPSIT introduces significant technical and operational challenges, including more complex wetting and venting procedures that may bring increased risk of contamination and susceptibility to human error.
F. Pre-use post-sterilization integrity testing (PUPSIT) has emerged as a critical standard in biopharmaceutical manufacturing, driven by the revised EU GMP Annex 2 and global regulatory harmonization.
G. The evidence does not state that pUPSIT aims to verify the integrity of sterilized filters before use, reducing the risks of defect masking and safeguarding drugs against contamination.
H. This review outlines regulatory requirements and risk-based rationales for the PUPSIT evaluation process and discusses the integration of PUPSIT within a broader contamination control strategy.


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 plastic pollution crisis urges innovative recycling solutions. Promising approaches especially for polyester-containing wastes include enzymatic hydrolysis and microbial upcycling. For efficient enzymatic hydrolysis of polyesters, elevated temperatures (70–80 °C) are required, necessitating thermophilic microbial chassis for consolidated bioprocessing (CBP). In this study, we engineered Geobacillus thermoleovorans through adaptive laboratory evolution (ALE) for robust growth on adipic acid (AA) and 1,4-butanediol (BDO), two relevant monomers for example derived from poly(butylene adipate- co -terephthalate) (PBAT), enabling growth rates of up to 0.10 h −1 on AA and 0.13 h −1 on BDO. Based on a high-quality annotated genome sequence of the wild type, genomic mutations and gene expression levels were characterized in mutants grown on the respective substrates compared to glucose. For BDO, an alcohol dehydrogenase (Gth_001044) and an aldehyde dehydrogenase (Gth_001082) were identified to be likely responsible for its oxidative degradation. AA uptake appears to be mediated by a dicarboxylate transporter (Gth_003270), followed by CoA activation and β-oxidation involving a CoA transferase (Gth_003192) and several upregulated CoA-family dehydrogenases. To demonstrate applicability of these strains in plastic upcycling, they were co-cultivated with PBAT as the sole carbon source in combination with the cutinase HiC for PBAT hydrolysis. This resulted in growth on the released AA and BDO. Given the potential to purify the remaining terephthalate (TA), this approach highlights the feasibility of selective monomer valorization in bioprocesses. Additional ALE enabled co-utilization of AA and BDO by a single strain and improved AA consumption at lower concentrations, underscoring the strains’ adaptability and high potential for plastic upcycling applications. • G. thermoleovorans evolved for robust growth on adipate and 1,4-butanediol at 60 °C. • Genome and transcriptome analyses revealed underlying pathways and enzymes involved. • Co-cultivation of the evolved strains on PBAT with HiC as the sole carbon source. The online version contains supplementary material available at 10.1007/s00253-026-13836-8.

Candidates:
A. For efficient enzymatic hydrolysis of polyesters, elevated temperatures (71–80 °C) are required, necessitating thermophilic microbial chassis for consolidated bioprocessing (CBP).
B. The evidence does not state that the plastic pollution crisis urges innovative recycling solutions.
C. The plastic pollution crisis urges innovative recycling solutions.
D. Promising approaches especially for polyester-containing wastes include enzymatic hydrolysis and microbial upcycling.
E. In this study, we engineered Geobacillus thermoleovorans through adaptive laboratory evolution (ALE) for robust growth on adipic acid (AA) and 2,4-butanediol (BDO), two relevant monomers for example derived from poly(butylene adipate- co -terephthalate) (PBAT), enabling growth rates of up to 0.10 h −1 on AA and 0.13 h −1 on BDO.
F. The evidence does not state that promising approaches especially for polyester-containing wastes include enzymatic hydrolysis and microbial upcycling.
G. For efficient enzymatic hydrolysis of polyesters, elevated temperatures (70–80 °C) are required, necessitating thermophilic microbial chassis for consolidated bioprocessing (CBP).
H. In this study, we engineered Geobacillus thermoleovorans through adaptive laboratory evolution (ALE) for robust growth on adipic acid (AA) and 1,4-butanediol (BDO), two relevant monomers for example derived from poly(butylene adipate- co -terephthalate) (PBAT), enabling growth rates of up to 0.10 h −1 on AA and 0.13 h −1 on BDO.


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: ["26 °C", "25 °C"]

Evidence:
The increasing production of biodiesel has led to a surplus of glycerol, a polluting by-product in need of valorization. In this study, we demonstrate that Citrobacter telavivensis T1.2D-1, an extremophile bacterium isolated from the Iberian Pyrite Belt, effectively converts glycerol into valuable compounds via dark anaerobic fermentation. Genomic and bioinformatic analyses confirmed the presence of the dha and pdu operons, responsible for 1,3-propanediol (1,3-PDO) synthesis, and the hyc operon and fdhF gene involved in hydrogen (H 2 ) production. Batch fermentations revealed that optimal yields of both H 2 (0.94 mol . mol-glycerol -1 ) and 1,3-PDO (0.66 mol‧mol-glycerol -1 ) were achieved at 25 °C using 2 g L -1 of supplied glycerol. Optimum yield of ethanol (1 mol‧mol-glycerol -1 ) was achieved using 12.5 g L -1 of supplied glycerol. Interestingly, 1,3-PDO and H 2 production inversely correlated with ethanol formation, suggesting metabolic competition. Antibiotic sensitivity profiling revealed susceptibility to multiple antibiotics, supporting future genetic engineering efforts. We suggest opperating with reactors at low concentrations to produce 1,3-PDO and H 2 with high yields, and at medium concentrations to generate ethanol. Our findings support C. telavivensis T1.2D-1 as a promising venue for the sustainable biotechnological production of biohydrogen and bio-based 1,3-PDO from glycerol, offering a dual solution to both energy demands and industrial waste 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:
Digital twins of mammalian cell cultures hold great potential for predictive bioprocess modeling, yet their development is challenged by the nonlinear dynamics and metabolic complexity of these systems. We present a hybrid computational framework that integrates mechanistic and data-driven modeling to construct predictive digital twins for Chinese hamster ovary (CHO) cell cultures producing monoclonal antibodies. The framework couples ordinary differential equation (ODE) models with constraint-based metabolic modeling and machine learning components trained on Bayesian-estimated metabolic rates. Applied to 23 CHO fed-batch cultures, viable cell density, product titer, and key metabolite concentrations are accurately predicted under varying feeding and media conditions within a unified simulation engine, where empirical variability is incorporated through multivariate statistical constraints derived from experimental data. Cross-validation analyses demonstrated strong generalization across process variations, highlighting the framework’s capacity to capture both biochemical constraints and adaptive cellular behavior. This hybrid modeling approach provides a mechanistically interpretable yet data-adaptive foundation for constructing bioprocess digital twins. By bridging statistical, mechanistic, and machine learning methodologies, it advances the computational representation of CHO cell culture systems and offers a generalizable strategy for predictive modeling in complex biological production processes.

Candidates:
A. We present a hybrid computational framework that integrates mechanistic and data-driven modeling to construct predictive digital twins for Chinese hamster ovary (CHO) cell cultures producing monoclonal antibodies.
B. Digital twins of mammalian cell cultures hold great potential for predictive bioprocess modeling, yet their development is not challenged by the nonlinear dynamics and metabolic complexity of these systems.
C. Digital twins of mammalian cell cultures hold great potential for predictive bioprocess modeling, yet their development is challenged by the nonlinear dynamics and metabolic complexity of these systems.
D. The evidence does not state that the framework couples ordinary differential equation (ODE) models with constraint-based metabolic modeling and machine learning components trained on Bayesian-estimated metabolic rates.
E. The evidence does not state that we present a hybrid computational framework that integrates mechanistic and data-driven modeling to construct predictive digital twins for Chinese hamster ovary (CHO) cell cultures producing monoclonal antibodies.
F. Applied to 24 CHO fed-batch cultures, viable cell density, product titer, and key metabolite concentrations are accurately predicted under varying feeding and media conditions within a unified simulation engine, where empirical variability is incorporated through multivariate statistical constraints derived from experimental data.
G. The framework couples ordinary differential equation (ODE) models with constraint-based metabolic modeling and machine learning components trained on Bayesian-estimated metabolic rates.
H. Applied to 23 CHO fed-batch cultures, viable cell density, product titer, and key metabolite concentrations are accurately predicted under varying feeding and media conditions within a unified simulation engine, where empirical variability is incorporated through multivariate statistical constraints derived from experimental data.


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:
Isobutanol-producing S. cerevisiae strain for industrial application. Isobutanol-tolerant strain developed from conventional mutagenesis. CRISPR-Cas9-mediated BAT1 knockout to enhance isobutanol production. CRISPR-Cas9 avoids the presence of plasmids and antibiotic markers. Strain highly increased isobutanol yield, potential for bio-alcohol production. Keywords: Saccharomyces cerevisiae , Isobutanol, Isobutanol toxicity tolerant, Mitochondrial Branched-chain amino acid aminotransferase Bat1, CRISPR/Cas9

Candidates:
A. The evidence does not state that cerevisiae strain for industrial application.
B. The evidence does not state that isobutanol-tolerant strain developed from conventional mutagenesis.
C. CRISPR-Cas9-mediated BAT1 knockout to enhance isobutanol production.
D. CRISPR-Cas10-mediated BAT1 knockout to enhance isobutanol production.
E. CRISPR-Cas10 avoids the presence of plasmids and antibiotic markers.
F. cerevisiae strain for industrial application.
G. Isobutanol-tolerant strain developed from conventional mutagenesis.
H. CRISPR-Cas9 avoids the presence of plasmids and antibiotic markers.


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 recombinant production of the protein-glutaminase (PG) from Bacteroides helcogenes (PGB) was investigated in the three Bacillus subtilis strains: 168, RIK1285 and the isolated strain 007. B. subtilis 007 produced the highest PG activity (7.6 ± 0.7 µkat L Culture supernatant −1 ) in shake flask cultivations and, thus, was used for further investigations. As a wild-type strain, B. subtilis 007 exhibited high extracellular proteolytic activity and formed large amounts of foam in a bioreactor cultivation. While the proteolytic activity would be favorable for the extracellular cleavage of PGB’s propeptide, foaming is undesirable and should be prevented. Therefore, the influence of the two foam-demolishing substances anti-foaming agent 204 and rapeseed oil on the PGB production was investigated in bioreactor cultivations. A PGB activity of 11.8 ± 1.1 µkat L Culture supernatant −1 was obtained using anti-foaming agent 204, while rapeseed oil led to a lower maximal PGB activity of 7.6 ± 0.2 µkat L Culture supernatant −1 . PGB was partially purified from the culture supernatant by fractionated ammonium sulphate precipitation followed by hydrophobic interaction chromatography. A final yield of 14.8% was obtained, while the specific PGB activity increased from 6.7 to 28.6 nkat mg −1 . Alternatively, PGB was purified from culture supernatant by cross-flow filtration and subsequent heating step (1 h, 60 °C) to inactivate the extracellular peptidases. Thereby, the total proteolytic activity was decreased to below 4% while the specific PGB activity increased to 32.1 ± 2.0 nkat mg −1 . The PGB preparation obtained was applied in gluten deamidation experiments which showed that PGB deamidated a 1% (w/v) gluten suspension by 95 ± 2%. The online version contains supplementary material available at 10.1007/s12010-026-05637-6.