Generational age-related Chinese hamster ovary (CHO) cell growth changes are undesirable for large-scale manufacturing cell lines. An age-related high growth phenotype in CHO cells has previously been shown to be associated with a Chromosome 2 (Chr2) duplication event in late generation cells. We have evaluated the contribution of a subset of the 123 genes involved in the duplication event in relation to the high growth phenotype of late generation cells (> 90 generations post-cloning). We show that this age correlated high growth phenotype associated with a Chr2 duplication event in CHO cells can be reversed by single knockdown of several transcripts that are upregulated as a result of the duplication event. Further, one of these genes, Cxxc1, when overexpressed in low growth phenotype cells, results in the generation of high growth phenotype cell pools. Thus, Cxxc1 overexpression engineering in CHO cells can impart a high growth phenotype that correlates with those observed in Chr2 duplication event cell lines. Furthermore, this impact can be reversed by subsequent knockdown of Cxxc1 transcript levels such that the cells revert to a low growth phenotype. The high growth phenotype of Cxxc1 overexpressing engineered cell pools was shown to be stable for 100 generations, and although the high growth phenotype was associated with a reduced cell specific productivity, volumetric titre was maintained/compensated for by the increased cell number. We propose targeted cell engineering of CHO cells to overexpress Cxxc1 may be a potential strategy to overcome the issue of age-related genetic instability associated growth phenotype to generate new host cell lines with a stable, high growth phenotype.
Bulk RNA-seq data suffers from the issues of “high dimensionality and small sample size,” which limits its application in disease research. This paper proposes a dynamic data augmentation method based on Layer-wise Relevance Propagation (LRP) aimed at improving classification performance and biological interpretability under small-sample conditions. The method utilizes the LRP algorithm to calculate the contribution weight of each gene to the classification result and uses this weight to guide sample generation. By systematically amplifying biologically meaningful signals, it constructs semantically reliable augmented samples, avoiding the semantic distortion caused by traditional random perturbations. Simultaneously, a dynamic augmentation mechanism is introduced that tightly couples sample generation with model training, providing difficult-to-classify samples with multiple iterative optimization opportunities and forming a virtuous cycle where classification performance and augmentation quality improve synergistically. On this basis, population-level gene biomarkers are identified from the trained model. Innovatively, an open-environment enrichment analysis method is proposed—that is, instead of being limited to a few feature genes of a single subtype, the union of feature genes from all disease subtypes is taken for enrichment analysis, revealing shared biological pathways from a systems-level perspective and providing a more comprehensive interpretation for subtype-specific mechanism research. Experimental results show that this method effectively improves classification accuracy, and through this open-environment enrichment approach, six hub genes were identified as gene markers for IgAN.
Wounds damage the skin, mucosal membranes, or underlying tissues, disrupting normal structure and function. Natural therapeutic agents, particularly plant extracts, have been widely used in wound management due to their antimicrobial and regenerative properties. Here, we report a novel multifunctional wound dressing by integrating Petroselinum crispum (parsley, PS) extract and zinc ascorbate-based metal-organic frameworks (MOFs) into a polyvinyl alcohol (PVA) nanofibrous matrix. The resulting MOF/PS-loaded PVA nanofibers (NFs) combine biodegradability, biocompatibility, and enhanced bioactivity for wound healing. The optimized formulation (PVA/MOF5/PS30-NFs) exhibited strong antibacterial activity, with inhibition zones of 250 mm², total phenolic content of 1.41 mg GAE/g, and antioxidant IC₅₀ values of 15.17 and 11.11 mg/3mL at 2 and 24 h, respectively. Cytocompatibility tests confirmed excellent cell viability (> 100
Polyphenols are plant-derived secondary metabolites with significant pharmaceutical potential, including flavonoids and stilbenoids. Microbial production of these compounds offers a sustainable alternative to plant extraction and chemical synthesis. However, most studies rely on glucose as a sole carbon source, limiting the use of lignocellulosic biomass, a sustainable and economical resource for biomanufacturing. Here, we present an engineering approach to develop a xylose-utilizing Saccharomyces cerevisiae platform that integrates carbon assimilation with plant polyphenol biosynthesis. We designed and optimized a naringenin biosynthetic module through heterologous gene screening, expression balancing, and culture optimization, achieving a 42-fold improvement (54.7 mg/L in XN03 vs. 1.3 mg/L in PSNR06) in production. This optimized module was subsequently extended to construct downstream pathways for resveratrol and apigenin biosynthesis, enabling the production of multiple polyphenols from xylose. Furthermore, we identified and implemented a previously uncharacterized flavone synthase from Artemisia argyi (AaFNS), which enabled highly efficient conversion of naringenin to apigenin with a molar conversion rate of 81
Pancreatic cancer remains a malignancy with limited treatment efficacy and high resistance to chemotherapy. miR-934 was identified as an oncogenic miRNA significantly upregulated in pancreatic cancer, promoting tumor progression, epithelial-mesenchymal transition (EMT), and chemoresistance by targeting the tumor suppressor PRUNE2. Inhibition of miR-934 sensitized cancer cells to Gemcitabine (GEM)-induced apoptosis, highlighting its therapeutic potential. To enhance delivery efficiency, AS1411 (oligonucleotide aptamer of nucleolin)-functionalized exosomes were engineered to co-deliver miR-934 inhibitor and GEM. The functionalized exosomes demonstrated high encapsulation efficiency, enhanced cellular uptake and tumor-targeting capability, and improved suppression of malignant phenotypes. This platform not only suppressed cell migration, invasion, and EMT but also enhanced GEM-induced apoptosis by restoring PRUNE2 expression and activating pro-apoptotic pathways. Furthermore, PRUNE2 overexpression reversed the malignant phenotypes and gemcitabine resistance induced by miR-934, further confirming the critical role of the miR-934/PRUNE2 axis in pancreatic cancer progression. In vivo, AS1411-functionalized engineered exosomes co-delivering miR-934 inhibitor and gemcitabine exhibited enhanced tumor accumulation and significantly suppressed tumor growth compared with free drug treatment and non-functionalized exosomes. These findings underscore the potential of aptamer-functionalized exosomes as a targeted therapeutic strategy for pancreatic cancer by integrating miRNA modulation and chemotherapeutic delivery.
Trichloroethylene (TCE), a known carcinogen, is denser than water, which explains its deep presence in groundwater. Limitations on long-term supplies of TCE degraders and nutrients often led to TCE dichlorination failures in the field. This study presents the development and evaluation of an agrowaste-based multilayer bioencapsulation system designed to prolong microbial activity and enhance nutrient availability for a more favorable TCE-dechlorination environment. TCE-degrading consortia were integrated with a nutrient-rich core derived from recycled fermented agrowastes and encapsulated within a structurally reinforced sodium alginate matrix to form bio-pellets. The acclimated microbial community exhibited a degradation rate constant of 0.33 day⁻¹, with confirmed transformation of TCE into dichloroethylene and vinyl chloride, indicating active reductive dechlorination pathways. Characterization of the encapsulated system revealed a nutrient profile of 410.4 ± 29.7 mg TOC g⁻¹, 9.1 ± 0.2 mg TN g⁻¹, and 20.5 ± 0.7 mg TP g⁻¹, which enabled sustained nutrient release to support microbial metabolic functions. Column reactor experiments demonstrated that the multilayer structure maintained physical integrity and functional stability for more than 74 days. The system achieved a total TCE removal efficiency of 98.5
3D bioprinting enables the layer-by-layer fabrication of living tissue constructs and supports patient-specific customization. This technology holds strong promises for regenerative medicine and drug discovery. However, its broader translation remains limited by material variability, safety considerations, cost constraints and regulatory requirements. This study investigates the use of safe, affordable and regulatory-compliant pharmaceutical polymers as biomaterials for 3D bioprinting. It specifically focuses on hydrogels formulated from Starch 1500®, maltodextrin and sodium alginate. The objective is to assess their potential applications in skin tissue engineering and oral drug delivery through semisolid extrusion-based 3D bioprinting techniques. Ionic crosslinking of the hydrogel was confirmed by FTIR analysis. The hydrogel exhibited a viscosity of 1.56 × 106 mPa·s, supporting semisolid extrusion bioprinting and excellent printability under ambient conditions. It enabled the fabrication of multilayer scaffolds with uniform filaments, well-defined square pore geometry and good shape fidelity. Rheological analysis showed shear-thinning behavior under applied stress, 87
Methane (CH4) is an attractive carbon feedstock for sustainable biomanufacturing, but poor CH4 solubility and gas-liquid mass-transfer limitations continue to constrain methanotroph-based CH4 valorization. Here, we evaluated biocompatible bionanofluids as additive-based process intensification tools and examined whether two operationally distinct bionanofluid application strategies produce distinct time-dependent physiological responses beyond simple growth enhancement. Two bionanofluids, chitosan/oleamide (CS/OA) nanoparticles and tannic acid–Fe3+ complex-coated cellulose nanocrystals (TA-Fe3+CNCs), were evaluated in 5-L cultures of Methylosinus trichosporium OB3b using two different application approaches: CS/OA seed adaptation followed by cultivation in conventional medium, and TA-Fe3+CNCs supplementation in the main culture. In both approaches, time-resolved profiling of gas uptake, nitrogen species, extracellular metabolites, and poly(3-hydroxybutyrate) (PHB) showed higher CH4 and O2 utilization, with 1.5–1.6- and 2.0–2.1-fold increases in specific CH4 and O2 uptake rates, respectively, and faster growth, with 1.3–1.5-fold higher µmax, than in the conventional control. These changes were accompanied by consistently lower PHB content, faster nitrate depletion, higher ammonium accumulation, and distinct extracellular metabolite patterns. Notably, CS/OA seed adaptation was associated with transient succinate accumulation during exponential growth, whereas TA-Fe3+CNCs supplementation led to faster methanol accumulation without succinate release. These observations are consistent with a process-level response in which higher CH4/O2 uptake and faster growth coincided with lower PHB content under the tested conditions. As an initial proof-of-concept experiment, a consecutive strategy combining CS/OA seed adaptation with TA-Fe3+CNCs supplementation in Methylomonas sp. DH-1 was associated with greater transient succinate accumulation and a higher peak succinate titer than under the conventional condition. Comparative time-resolved profiling of two different bionanofluid approaches indicated that bionanofluid application was associated with higher CH4/O2 uptake, faster growth, lower PHB accumulation, and distinct time-dependent metabolite patterns under the tested conditions. These findings support the use of biocompatible bionanofluids as practical media additives that can be applied without major bioreactor redesign and can motivate future component-controlled testing of sequential strategies for growth-linked CH4 bioconversion.
Functional small-diameter tissue-engineered vascular grafts (TEVGs) require seed cells with robust proliferation and extracellular matrix (ECM) synthetic capacity. Human induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) are promising but often functionally immature. While physiological hypoxia is a key developmental signal, its effects on hiPSC-VSMC phenotype, metabolism, and redox homeostasis remain unclear, and whether hypoxia-preconditioned cells can facilitate scaffold-free TEVG fabrication is unexplored. HiPSCs were differentiated into VSMCs under normoxic or hypoxic conditions. Integrated transcriptomic, metabolomic, and functional assays were employed to evaluate phenotypic, metabolic, and redox changes. Peroxisome proliferator-activated receptor alpha (PPARα) was identified as a potential central hub by bioinformatics and its activity was modulated using agonist (WY14643) and antagonist (GW6471). A cell sheet (CS) platform was established and optimized by screening medium additives and seeding density. Optimized CSs were then rolled and matured within a bioreactor to generate scaffold-free TEVGs, followed by decellularization to produce ECM scaffolds evaluated for decellularization efficiency and ECM integrity. Physiological hypoxia (5
The global transition toward a sustainable bioeconomy requires efficient microbial platforms for converting renewable feedstocks into energy-dense chemicals. Yarrowia lipolytica is a well-established oleaginous yeast used in lipid biotechnology because of its acetyl-CoA metabolism and genetic tractability. However, lipid biosynthesis is frequently limited by restricted intracellular NADPH availability, creating a common redox bottleneck. To address this, we investigated the use of formate as an auxiliary electron donor in combination with heterologous NADP+-dependent formate dehydrogenase (FDH) and lipid-pathway engineering. We overexpressed the highly NADP⁺-specific formate dehydrogenase (FDH) from Pseudomonas sp. 101 and observed an increase of up to 47.8
Tuberculosis (TB) remains a leading global health challenge, accounting for approximately 1.4 million deaths annually and disproportionately affecting low-and middle-income countries. Despite advances in diagnostics, rapid and affordable TB detection remains limited in resource-constrained settings. Culture-based methods, while highly sensitive, require prolonged incubation, and sputum smear microscopy, though widely used, is low in sensitivity, particularly at low bacterial loads. Molecular assays such as GeneXpert offer improved sensitivity but are costly and infrastructure-dependent, restricting their accessibility in high-burden regions. This study developed a rapid, stepwise, low-cost diagnostic workflow that integrates glycan-functionalized magnetic nanoparticles (gMNPs) for mycobacterial isolation and enrichment with a gold nanoparticle (GNP)-based plasmonic biosensor for molecular specificity. Using Mycobacterium smegmatis (Msm) as a validated biosafety level 1 surrogate for Mycobacterium tuberculosis (Mtb), gMNPs were optimized to capture and concentrate mycobacteria from liquid suspension and Ziehl-Neelsen visualization. Following magnetic enrichment and DNA extraction, plasmonic biosensing was performed using an IS6110-targeting TB probe and an Msm-specific probe. Analytical sensitivity and selectivity were assessed using absorbance ratio measurements (A520/A620), serial DNA dilutions, and comparisons against non-mycobacterial respiratory species, including Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, Streptococcus spp., Escherichia coli, and Staphylococcus aureus. Magnetic enrichment significantly increased bacterial recovery, extending the analytical detection range and enabling visualization of acid-fast bacilli (AFB) as low as 10² CFU/mL, which are otherwise undetectable by conventional smear microscopy. The plasmonic biosensor successfully differentiated mycobacterial samples from non-mycobacterial controls without PCR amplification. Statistically supported limits of detection were determined as 2.5 ng/µL for the Msm-specific probe and 5 ng/µL for the IS6110-targeting TB probe. Sequence alignment demonstrated 87
Glial scar formation and neuronal loss following spinal cord injury (SCI) are major impediments to neural regeneration. Therefore, remodeling the inhibitory scar obstacles and replenishing lost neurons are paramount for neural repair and functional recovery. However, current prevailing therapeutic approaches faced formidable challenges in concurrently achieving effective neuronal replenishment and glial scar (GS) remodeling. Herein, we described an innovative “remodeling-regeneration integrated” non-viral therapeutic strategy designed to directly reprogram reactive astrocytes within GS into functional neurons of distinct subtypes in vitro. This reprogramming is achieved by utilizing poly(β-amino ester) (PBAE) nanoparticles for the efficient co-delivery of the proneural transcription factors ASCL1 and NGN2 (TFs-A/N) into glial scar cells. For the delivery of two genes, the PBAE polymer was synthesized under an optimized Michael addition reaction condition. Our results demonstrated that this polymer possesses low toxicity, favorable biodegradability, and high molecular weight, allowing efficient encapsulation of substantial amounts of TFs-A/N-encoding plasmids into stable nanoparticles. Importantly, these optimized nanoparticles exhibited high gene delivery efficiency. Of particular significance, PBAE-TFs-A/N nanoparticles not only effectively remodeled GS architecture but also induced the generation of diverse neuronal subtypes, including cholinergic, glutamatergic, GABAergic, and dopaminergic neurons, as evidenced by subtype-specific biochemical marker expression. Furthermore, the converted cells acquired neuronal functionality, demonstrated by expression of synaptic markers (SYN and PSD95), action potential firing, and neuron-specific sodium and potassium currents. These findings provide compelling evidence for a safe and effective non-viral reprogramming methodology that simultaneously promotes multi-subtype neuron generation and GS remodeling. Collectively, the study positions the approach as a promising avenue for novel insights into CNS injury repair and clinical translational applications.
Radiation-induced skeletal deterioration and bone loss remain major clinical challenges. Although exosomes derived from human bone marrow-derived mesenchymal stromal cells (hBMSCs) (MSC-Exos) are promising cell-free therapeutics, their limited stability and low delivery efficiency hinder translational application. Here, we developed an innovative microdroplet-based exosome delivery platform (MD/Exos) and systematically evaluated its protective effects under irradiation-induced stress. MSC-Exos were successfully isolated and characterized using transmission electron microscopy and western blot analysis. Fluorescence imaging and confocal microscopy confirmed efficient uptake of MSC-Exos by both macrophages and hBMSCs. MD/Exos were fabricated by encapsulating exosomes within bovine serum albumin-stabilized microdroplets. Cytocompatibility assays showed that MD/Exos exhibited no detectable cytotoxicity toward macrophages. Functionally, MD/Exos significantly enhanced the viability, metabolic activity, and cytoskeletal integrity of irradiated hBMSCs. Notably, MD/Exos markedly promoted osteogenic differentiation after irradiation, as evidenced by increased alkaline phosphatase activity, enhanced mineralized matrix deposition, and upregulated osteogenesis-related gene expression. In parallel, MD/Exos modulated macrophage responses to radiation by suppressing aberrant multinucleated giant-cell formation and reducing pro-inflammatory cytokine expression. In vivo, administration of MD/Exos in an irradiation-induced bone injury model significantly attenuated bone loss and promoted bone regeneration, as evidenced by preserved bone microstructure, reduced osteoclast activity, and downregulated tumor necrosis factor-alpha (TNF-α) expression. Further in vivo cytokine analysis indicated that MD/Exos treatment significantly reduced pro-inflammatory cytokines compared to irradiated controls. Collectively, these findings demonstrate that MD/Exos represent a biocompatible and efficient exosome delivery platform capable of mitigating irradiation-induced cellular dysfunction, enhancing osteogenesis, and modulating immune-bone crosstalk. This study highlights the therapeutic potential of MD/Exos as a novel strategy for the prevention and treatment of radiation-induced bone loss.
The development of smart natural delivery systems that can deliver several chemotherapeutics in a controlled manner is a promising idea to enhance cancer treatment efficacy. In this study, we designed and synthesized a smart, dual pH- and thermo-responsive nanocarrier system based on β-CD-g-BCN, which was further functionalized with MSP and copolymerized with NIPAAm and DMAEMA to yield the final β-CD-g-BCN@MSP@PND formulation. The nanocarriers indicated efficient encapsulation of two anticancer agents, MTX and CUR, with encapsulation efficiencies of 64.5
Platelet-rich plasma (PRP) is widely used in regenerative medicine due to its high content of growth factors and bioactive molecules involved in tissue repair and regeneration. However, the lack of standardized preparation protocols leads to significant variability in clinical outcomes. To address this limitation, photobiomodulation (PBM) has emerged as a promising alternative to conventional chemical activation methods, enabling more controlled PRP stimulation and sustained growth factor release, while avoiding the rapid burst secretion and potential platelet damage induced by thrombin or CaCl₂. In this study, we developed a compact illumination system capable of delivering fluence-controlled irradiation at 660 and 830 nm for ex vivo PRP activation and evaluated its effects using an integrated analytical approach combining ELISA and Raman spectroscopy. The effects of different fluences (10–15 J/cm2) and incubation times (1, 24, 48 and 72 h) were systematically investigated to evaluate dose–response effects on PRP activation. To support real-time, label-free monitoring and facilitate clinical translation, Raman spectroscopy was assessed as a non-destructive analytical tool, with ELISA used for reference validation. Our results show that PBM induces reproducible biochemical changes in PRP, with Raman spectral signatures at 72 h (10 and 15 J/cm²) correlating with ELISA growth factor release. This study demonstrates that controlled light-based stimulation enables tunable PRP activation while preserving platelet structural integrity. Integrating a compact PBM device with portable Raman spectroscopy provides a non-destructive, clinically compatible strategy for both modulating and monitoring PRP activation. This approach establishes a technological framework for standardized, dose-defined PRP photoactivation and supports the development of translational, light-guided regenerative therapies.
Activatable antibodies aim to minimize on-target off-tumor toxicity by suppressing antigen binding through a masking domain that is released only upon encountering a tumor-associated stimulus. Anti-idiotypic single-domain antibodies (dAbs) raised against the complementarity-determining regions (CDRs) of a parental antibody are attractive masking modules because they engage the paratope through a native, structurally defined antibody–antibody interface. However, the high intrinsic affinity that makes such dAbs effective masks can also prevent full recovery of antigen binding after protease-mediated cleavage of the linker connecting the mask and the antibody. Here we addressed this limitation by structure-guided affinity tuning of an anti-trastuzumab dAb mask fused to the anti-HER2 4D5 single-chain variable fragment (4D5scFv). Analysis of the dAb–trastuzumab crystal structure (PDB ID 7PKL) identified Y61 and W104 of the dAb as principal anchor residues at the masking interface. An initial alanine scan showed that the wild-type construct effectively suppressed HER2 binding prior to cleavage but failed to recover binding after Tobacco etch virus (TEV) protease cleavage, whereas the single mutants Y61A and W104A restored full unmasking. The Y61A/W104A double mutant lost masking entirely. Fine-tuning at the more extensively engaged W104 position (W104A, W104L, W104F) identified W104F as the optimal variant, yielding the largest masked-to-unmasked dynamic range of approximately 22-fold, calculated as the ratio of the EC50 value of uncleaved W104F to that of TEV-cleaved W104F, while preserving complete recovery of antigen binding. Replacement of the TEV site with a matrix metalloproteinase-9 (MMP-9) cleavage site preserved this behavior in a tumor-relevant context: dAb(W104F)-MMP-9-4D5scFv exhibited approximately 125-fold suppression of apparent HER2-binding potency in the absence of MMP-9 and recovered binding to within approximately 3-fold of the parental 4D5scFv after cleavage. BLI analysis further showed detectable HER2 binding of MMP-9-treated dAb(W104F)-MMP-9-4D5scFv, with an apparent KD of 10.0 nM, whereas dAb(WT)-MMP-9-4D5scFv and uncleaved dAb(W104F)-MMP-9-4D5scFv showed no detectable binding under the same assay conditions. In a cell-based binding assay on HER2-positive BT-474 cells, only the W104F construct displayed clear MMP-9-dependent cellular binding, while the wild-type construct remained inactive under both conditions. Notably, MMP-9-dependent cleavage was also confirmed under a 10
Cutaneous oncology needs to be treated locally with maximal exposure to the target with minimum systemic toxicity. Novasomes, surfactant-rich vesicles, have proven to be promising theranostic carriers for both topical and intralesional delivery. This review aims to establish a translational framework linking novasome design with theranostic-guided dosing in cutaneous oncology. A literature search of publications from the time period 2010–2026 was conducted in Scopus, PubMed, and Web of Science to capture studies on formulation, in vitro/ex vivo penetration data, in vivo efficacy, and biodistribution, and the first clinical reports; the priority was given to studies with orthogonal imaging, microdialysis, and/ or IND-relevant endpoints. The literature shows that novasomes allow improved local drug retention, controllable release kinetics, and better follicular and tumor penetration than traditional topical systems. Surface modification and deformable bilayers also help further optimise targeting, and co-loading imaging agents enables real-time visualisation, image-guided dosing, and adaptive PK/PD modelling. Advances in scalable fabrication techniques such as microfluidics and solvent-minimized processes enhance translational feasibility. However, there are still challenges in long-term stability, standardized reporting of critical quality attributes, and regulatory harmonization. In conclusion, novasomes are a pragmatic and modular platform for localized image-guided cutaneous oncology. A unique aspect of this review is the inclusion of novasome formulation strategies, theranostic functions, and concepts of PK/PD-guided dosing in a translational context for cutaneous oncology. The review bridges the gap between formulation development and clinical implementation aspects, thereby offering a practical view for future precision nanomedicine strategies.
Monoclonal antibody (mAb) therapies are limited by factors affecting their pharmacokinetics (PK), such as anti-idiotypic antibodies (anti-ID Abs) and the antigen sink effect. To address this, we developed six pro-anti-TNFα Abs (pro-Infliximab) incorporating unique hinge regions (“Ab locks”) from different immunoglobulin subtypes linked via protease-sensitive peptides. The steric shielding formed by the endogenous hinge’s disulfide bonds provides a modular design that can be applied to various Ab drugs with minimal structural modification. Compared to other spatial hindrance strategies, this Ab lock approach is anticipated to offer a favorable immunogenicity profile. Among the variants, The IgG1 hinge exhibited the most robust masking capability among the evaluated domains, resulting in a 128-fold reduction in Infliximab’s antigen binding. IgG1 pro-Infliximab demonstrated low immunogenicity after repeated dosing and maintained stable PK in both naïve and immunized mice, retaining 74.2
The human microbiome provides a robust ecosystem for mucosal barrier function, immune regulation, metabolism, and resistance to pathogen infection. Dysbiosis causes inflammatory bowel disease, oral inflammation, wound damage, skin disease, and cancer-related immune dysregulation. Current probiotic therapy is limited by low survival during storage and administration, low oral bioavailability, short retention time, and insufficient targeting. Probiotic-hydrogel systems combine viable microorganisms with natural or synthetic polymer networks to promote probiotic protection, local retention, and controlled release. They can also incorporate prebiotics, engineered bacteria, oxygen-releasing agents, antioxidants, growth factors, drugs, or advanced manufacturing techniques such as microfluidics and 3D printing. Recent preclinical studies suggest that probiotic-hydrogel can modulate local microbes, suppress pathogens, control inflammation, support barrier repair, and promote tissue regeneration. However, most evidence is far from clinical, and human data are limited to a small number of oral and periodontal applications. In this review, we summarize the design strategies, encapsulation approaches, mechanistic frameworks, disease-based evidence, and translational challenges of probiotic-hydrogel. In particular, we paid attention to evidence level, safety, manufacturing, regulatory classification, product stability, clinical trial design. Such issues need to be well understood before probiotic-hydrogel can move from experimental platforms to clinically applicable microbiome treatments.
Terpenoids are attractive targets for sustainable biomanufacturing because of their structural diversity and potential applications in fuels and high-value chemicals. Among sesquiterpenes, γ-curcumene is a promising yet underexplored molecule, and its efficient microbial production has not been established. In this study, we identified a previously uncharacterized γ-curcumene synthase PaCS from Parthenium argentatum and evaluated its use for microbial γ-curcumene production in metabolically engineered Escherichia coli. Sequence analysis revealed that PaCS is phylogenetically distinct from the previously reported γ-curcumene synthase from Pogostemon cablin. When expressed in an E. coli strain carrying a heterologous mevalonate pathway, PaCS predominantly produced γ-curcumene, with only minor formation of (−)-α-bisabolol. Product identity was confirmed by GC–MS and NMR analyses. Biochemical characterization of recombinant PaCS showed maximal activity at 35 °C and pH 8.5, with Mg²⁺ as the preferred divalent metal ion. For microbial production, culture conditions and host background were evaluated, and the evolved strain SBA01 showed the best performance. In fed-batch fermentation, the engineered strain produced up to 1.1 g/L γ-curcumene in terrific broth and 1.03 g/L in modified R medium. In addition, purified γ-curcumene was chemically hydrogenated to bisabolane and dihydro-ar-curcumene, demonstrating its utility as a precursor for fuel-related sesquiterpene hydrocarbons. This study establishes a microbial production platform for γ-curcumene using a novel γ-curcumene synthase from P. argentatum and metabolically engineered E. coli. The results expand the available terpene synthase toolbox and provide a foundation for the biosynthesis and downstream upgrading of γ-curcumene-derived molecules. These findings support the broader use of synthetic biology and metabolic engineering for the production of non-native sesquiterpenes from renewable carbon sources.
