Recombinant adeno-associated virus (rAAV) has shown great promise as a viral vector for gene therapy. However, efficient manufacture of high-quality rAAV to meet clinical demands remains challenging. Here, we optimized rAAV production via a design of experiments (DoE) approach to evaluate the effects of five transfection parameters on vector genome titer, full particle ratio (FE ratio), and viral protein (VP) stoichiometry. The DoE model showed that no single set of transfection conditions could maximize all three responses simultaneously. Subsequently, the materials from DoE-optimized transfections were purified by affinity chromatography followed by anion exchange chromatography (AEX). We observed that AEX recovery declined when capsid loading was high, a situation caused by the combination of high titer with low FE ratio, reducing some of the gains in titer achieved by transfection optimization. Furthermore, AEX had limited capacity to enrich full particles from materials with a very low initial FE ratio. Finally, materials from DoE-optimized transfections showed improved transduction efficiency, which was associated with the ratios of VP1 and VP2 to total VP in the capsid. Overall, this study demonstrates that transfection-derived quality attributes affect purification outcomes and overall vector quality. Our findings highlight the necessity of strategically balancing multiple quality attributes during transfection optimization and provide insights for integrated upstream transfection and downstream purification development.

The basidiomycetous yeast Pseudozyma antarctica produces the biodegradable plastic-degrading enzyme PaE. The PaE overexpression strain, constructed by replacing the promoter of the PaE gene with the xylanase promoter, exhibited high-level production of PaE in the presence of xylose. However, this production is suppressed by carbon catabolite repression (CCR) in the presence of glucose. Although CCR is a major issue involving the decreased expression of hydrolytic genes from their promoters in ascomycetes, little information is available in basidiomycetous yeasts. In the present study, we demonstrated that glucokinase is involved in the regulation of CCR in P. antarctica. Ultraviolet irradiation of P. antarctica resulted in mutant strains that exhibited resistance to the toxic glucose analog 2-deoxyglucose and reduced the CCR of the xylanase and PaE driven by their own promoters. These strains had missense or frameshift mutation within the glucokinase gene (PaGLK1). The restoration of CCR through intact PaGLK1 complementation in these strains indicated that loss-of-function mutations in PaGLK1 contribute to CCR defects. Deletion of PaGLK1 in the PaE overexpression strain showed only a limited reduction in PaE production, even when 3% of the 4% xylose in the liquid medium was replaced with glucose. These results indicate that disruption of the glucokinase gene considerably reduces the cost of the PaE-producing medium and facilitates the efficient production of useful proteins in basidiomycetous yeasts.

A koji biocatalyst with chlorogenate hydrolase (CHase, EC 3.1.1.42) activity was produced by solid-state fermentation of Aspergillus niger AKU 3302 using yerba mate processing residues as both support and inducer. The fungus was cultivated in traditional koji trays until complete mycelial colonization. Thermal treatment of the koji at 60 °C for 30 min effectively inactivated fungal viability while preserving CHase activity and yielded a non-viable but highly active biocatalyst (YMKnon-viable). Maximum CHase activity of the biocatalyst (approximately 31 EU/100 gdry material) was obtained in a solid culture medium composed of discarded yerba mate powder supplemented with 7 g/L sucrose and 0.4% (w/v, dry basis) yerba mate extract after 4 days of fermentation. The YMKnon-viable exhibited good operational stability during two consecutive reaction cycles (up to approximately 330 min of continuous operation) and excellent storage stability, retaining 100% of its initial activity after 7 months at -20 °C. These results demonstrated that yerba mate processing residues could be efficiently valorized to produce a heat-inactivated, reusable CHase biocatalyst with good operational and storage stability, providing a simple and sustainable approach for the biotransformation of chlorogenic acid-rich extracts.

Benzotriazole ultraviolet stabilizers (BUVSs) are widely used as additives in a variety of materials to protect against UV-induced degradation. Their widespread distribution in environmental and biological samples has raised concerns regarding their potential ecological and health risks. 2-(2'-Hydroxyphenyl)benzotriazole (UV-P), a structurally simple BUVS, was selected as a model compound to evaluate its metabolism by the white-rot fungus Phanerochaete sordida in potato dextrose broth cultures. UV-P gradually decreased, with approximately 10% and 40% removed in 2 and 4 weeks, respectively. Two metabolites were identified as glucosylated and O-methylated derivatives of UV-P. These metabolites retained, and in some assays slightly increased, cytotoxicity relative to the parent compound. This finding demonstrates that fungal transformation of BUVSs can generate biologically active glycosylated or O-methylated products, emphasizing the need to identify transformation products rather than evaluating only parent-compound disappearance.

Extracellular environments regulate cell proliferation, differentiation, and function through dynamic interactions between cells and the extracellular matrix (ECM), as well as through soluble biochemical cues such as growth factors, all of which are governed by coupled biochemical reactions and mass transport processes. To engineer such environments in a controllable manner, we developed azide-cyclooctyne clickable alginate microcapsules that allow bioactive peptides to be introduced by in situ conjugation via strain-promoted azide-alkyne cycloaddition (SPAAC). Osteoblastic cells were encapsulated in cyclooctyne-modified alginate microcapsules, and branched bone morphogenetic protein 2 (BMP-2) mimetic peptides bearing azide groups were added to the culture medium. The peptides diffused into the microcapsules and selectively reacted with cyclooctyne groups without nonspecific reactions, resulting in spatially confined accumulation controlled by diffusion and click reaction kinetics. This localized enrichment of osteogenic signals promoted early osteogenic differentiation of the encapsulated cells, as indicated by activation of the Col1a1-GFP reporter. From a bioreaction engineering perspective, the present system demonstrates an in situ conjugation strategy for spatiotemporal regulation of bioactive molecules within microstructured materials, enabling modulation of early osteogenic responses without direct chemical modification of cells. This bioinspired in situ conjugation approach based on the click chemistry provides a versatile platform for biomedical, bioindustrial, and biochemical applications.

Pediocin, produced by certain lactic acid bacteria, has high antibacterial activity against Listeria monocytogenes and is therefore expected to be used as a peptide food preservative. In this study, a rapid and efficient method for recovering pediocin with antibacterial activity from a fusion protein with green fluorescent protein (GFP) produced by Escherichia coli was developed. After cell lysis, the concentrated fusion protein contained in the insoluble fraction was solubilized and desalted, and pediocin was cleaved from GFP by a protease factor Xa treatment. As pediocin exhibited strong adsorption to various membranes and a column matrix, and was presumed to adsorb to GFP, it was difficult to efficiently collect the pediocin with column purification and filtrations including ultrafiltration. Heating the mixture in boiling water was found to be an effective method to selectively remove GFP followed by centrifugation. Because pediocin is highly thermostable, soluble pediocin with high antilisterial activity was retained in the recovered supernatant. This study demonstrates that recombinant pediocin can be recovered rapidly and efficiently by the heat denaturation of cleaved fusion partner and centrifugation.

Postoperative adhesions occur frequently after abdominal surgery and gynecological surgeries, causing serious complications. For applications in laparoscopic surgery, we developed a thermally cross-linked gelatin film-cut (GC) by dividing a thermally cross-linked gelatin film (GF) into small fragments. In this study, we examined the basic biological properties of the GC and conducted anti-adhesion tests using a rat cecum abrasion model to evaluate the effects of film fragmentation, focusing on the influence of fragment size, dosage and film thickness. Solubility test results showed that GC degraded more slowly than its film counterparts. Furthermore, as the thickness and the size of the GC increased, its degradation rate decreased. In addition, the results of the collagenase degradation test demonstrated that the GC degraded more slowly than the unfragmented film and that as the thickness and size of the GC increased, its degradation rate decreased. The results of the rat anti-adhesion test demonstrated that compared with GF, GC was significantly less effective in preventing adhesion. However, compared with the untreated group, several GCs showed significantly lower adhesion scores. Some of the GCs demonstrated anti-adhesion effect similar to or better than those of hyaluronic acid and carboxymethyl cellulose (HA/CMC). In summary, increased fragment size, reduced film thickness, and an elevated dosage of GC improved anti-adhesion efficacy. Thus, GC exhibits excellent anti-adhesion effects when its geometry, thickness, and dosage are optimized. Consequently, anti-adhesive GC improves the clinical applicability of anti-adhesion materials, particularly in laparoscopic surgery.

Migrasomes are extracellular vesicles secreted by migrating cells that mediate intercellular communication and contribute to inflammation and cancer progression. We have previously demonstrated that IL-6 expression is induced in cells that have taken up migrasomes derived from IL-6 amplifier activated cells, suggesting that migrasomes are involved in the propagation of inflammation. However, the molecular changes occurring within migrasomes upon IL-6 amplifier remain unknown. In this study, migrasome-enriched samples were extracted from IL-6 amplification-induced human breast cancer MDA-MB-231 cells by ultracentrifugation, and comparative proteomics analysis was performed. Shotgun proteomics detected a total of 1984 proteins, of which 1339 were detected under all conditions. In the cytokine-stimulated migrasomes, 330 proteins up-regulated and 149 proteins down-regulated compared to starvation condition. Changes associated with IL-6 amplification included the IL-6 pathway kinase JAK1, proteins involved in vesicular transport, and SNARE family members syntaxin-3 and syntaxin-4. After immunostaining, the fluorescence of JAK1 in migrasomes under stimulation condition was approximately 1.5-fold higher than those under starvation condition, consistent with the proteomic result. Furthermore, functional enrichment analysis revealed enhanced expression of pathways related to oxidative phosphorylation, endoplasmic reticulum protein processing, and cell organization. These findings indicate that IL-6 amplifier activation is associated with coordinated changes in migrasome proteins, including those involved in inflammatory signaling and trafficking. This study provides a proteomic understanding of migrasome-mediated inflammatory communication in the tumor microenvironment.

Disease-suppressive soils, in which soilborne pathogens are naturally suppressed, offer a promising model for sustainable crop protection, particularly in organic farming systems where chemical disease control options are limited. Although disease suppression in these soils is considered to rely on biological control, the underlying mechanisms remain poorly understood. In this study, we investigated soil from a long-term organically managed field in Shiga Prefecture, Japan, where soilborne disease incidence has remained consistently low, to identify bacterial community features potentially associated with this field. The 16S rRNA gene amplicon sequencing indicated that this soil harbored a bacterial community distinct from those of nearby agricultural soils. Following the application of organic compounds, the genus Lysobacter, a taxon with known antagonistic activity against plant pathogens, was markedly enriched in response to proteinaceous organic inputs. This enrichment was consistent across sampling times and specific to certain proteinaceous organic inputs, whereas minimal effects were observed on chitin, N-acetyl-d-glucosamine, or cysteine. Broader soil surveys indicated that Lysobacter enrichment was not strictly associated with whether soils had been managed under organic or conventional farming practices. Stepwise multiple regression analysis identified 10 co-occurring bacterial genera that were strongly associated with Lysobacter abundance. These findings highlight condition-dependent Lysobacter enrichment as a characteristic microbial response to proteinaceous organic amendments in this low-disease-incidence field and provide microbial insights that may inform microbiome-based strategies for sustainable soil management.

Green Awak banana (Musa paradisiaca cv. Awak) is a tropical fruit widely cultivated in Indonesia and valued for its high resistant starch content, which contributes to improved glycemic control, gut health, and potential prebiotic effects. The growing prevalence of gluten intolerance and celiac disease has increased demand for naturally gluten-free alternatives, positioning banana flour as a promising substitute for wheat-based products. This study evaluated the influence of different drying techniques, including sun-drying, oven-drying, and freeze-drying, on the metabolomic profile, functional properties, and available carbohydrate of green Awak banana. Metabolite profiling was conducted using gas chromatography mass spectrometry (GC-MS), while multivariate analysis and in vitro available carbohydrate assays were performed to assess biochemical variation among drying treatments. GC-MS metabolite profiling annotated 79 metabolites, and multivariate analysis showed clear separation among drying treatments. Pathway enrichment highlighted starch and sucrose metabolism as a primary pathway affected by drying. Sun-dried samples exhibited the highest available carbohydrate despite lower free glucose intensity, indicating preservation of enzyme-accessible polymeric carbohydrates. In contrast, oven-dried samples showed higher low-molecular-weight sugars but reduced available carbohydrate, likely due to thermal modification. These results demonstrate that drying methods alter carbohydrate functionality rather than total carbohydrate content, supporting the potential of sun-dried green Awak banana as a functional, slowly digestible carbohydrate ingredient.

Diazotrophic l-glutamate (L-Glu) fermentation, independent of the Haber-Bosch process, is a sustainable strategy. Following our successful production of L-Glu from aerial nitrogen by an engineered Klebsiella pasteurii NG13 overproducing citrate synthase and citrate transporters (+CC strain), we herein examined L-Glu transporters in this bacterium. Extracellular L-Glu accumulation was abolished in a K. pasteurii strain in which a gene homologous to Escherichia coli ybjL was deleted, suggesting that the YbjL homologous protein (herein designated as GluE) was the main exporter of the +CC strain for diazotrophic L-Glu production. However, the overexpression of gluE did not increase diazotrophic L-Glu production, suggesting that the export of L-Glu was not a rate-limiting step in diazotrophic L-Glu production in the +CC strain. To sustain and increase diazotrophic L-Glu production, we constructed a triple knockout K. pasteurii +CC strain lacking the putative gltS, gltP, and gltIJKL orthologs, all the counterparts of which encode L-Glu importers in E. coli. In glucose-fed cultures, while L-Glu produced extracellularly by the +CC strain decreased during a long-term cultivation, the triple knockout strain sustained L-Glu production over the culture period and finally reached 2.67 g L[-1]. These results suggest the potential of the practical application of our system to the production of nitrogen-containing compounds from aerial nitrogen.

Sago starch is an important carbohydrate resource with a potential relevance for future food security. Flavor characteristics of sago starch play a critical role in product acceptability. To date, the influence of the milling grade and contribution of varietal differences of raw materials on its flavor characteristics, which are produced using similar processing methods, has not been sufficiently explored. In this study, we investigated the effects of varietal differences and milling grades on the sensory and metabolites profile of sago starch samples produced from two different sago varieties that have morphological differences, Tuni variety (Metroxylon rumphii) (spiny) and Molat variety (Metroxylon sagu Rottb.) (spinless), and three different milling process: local grade (milled using rotary shifter 100 mesh size), consumer grade (milled using rotary shifter 120 mesh size) and export grade (milled using vibrating screen 120 mesh size). The analyses were performed through integrated metabolomics and sensory quantitative descriptive analysis. Under similar production methods, varietal differences exerted a stronger influence than the milling grade on both the sensory and metabolite profiles of sago starch samples. Non-volatile metabolites were largely unaffected by the milling grade, whereas volatile compounds exhibited greater sensitivity to milling differences. Partial least squares regression analysis further revealed that sugars and specific volatile metabolites contributed to desirable and off-flavor characteristics between the two varieties. Overall, these findings provide the preliminary insight that varietal differences are the primary determinants of the flavor characteristics of sago starch, rather than milling grades, when the sago starch samples are produced using similar processing methods.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

The iminosugar 1-deoxynojirimycin (1-DNJ) is a pharmaceutically significant natural alkaloid with expanding therapeutic and nutraceutical applications. Its microbial biosynthesis offers a sustainable and scalable alternative to plant extraction. However, process optimization is constrained by the complex, non-linear interactions between nutritional and physicochemical fermentation parameters. Herein, the study aimed to enhance the production of 1-DNJ by Bacillus amyloliquefaciens using a combination of single-factor experiments and artificial neural network (ANN) models. LC-MS/MS analysis confirmed the strain produced 1-DNJ from soluble starch and peptone. A strategy combining single-factor experiments and an ANN machine learning model was then used to enhance 1-DNJ yield. The culture medium obtained through single-factor optimisation had α-glucosidase inhibitory activity of 82.55% and a 1-DNJ titer of 1064 ± 4.64 mg/L. A computationally efficient ANN, constructed with a weighted resilient backpropagation algorithm (rprop+) and six hidden neurons, was then trained on this limited dataset. The model identified refined fermentation conditions, predicting a further increase to 87.47% inhibition and a titer of 1179 ± 8.44 mg/L. Sensitivity analysis of the validated ANN pinpointed inoculum volume and fermentation temperature as the most critical process variables. This machine learning framework is a viable technical approach for optimising 1-DNJ fermentation. The identified key fermentation parameters lay a solid foundation for subsequent research.