Robust Production Research Articles

A modular platform for bioluminescent RNA tracking

AbstractA complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs. The RNA tags are engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production is observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag is necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

Efficient Neutralizing Antibodies Induction by Human Parvovirus B19 Epitope-Presenting Protein Nanoparticles.

Human parvovirus B19 (B19V) causes fetal hydrops in pregnant women. Despite the significant impact of this virus, effective vaccines remain unclear. In this study, we successfully engineered B19V protein nanoparticles by fusing the N-terminal receptor-binding domain corresponding to 5-80 amino acids of VP1 with two distinct types of self-assembling protein nanoparticles. Gel filtration and electron microscopic analysis confirmed the spherical assembly of the antigen-fused nanoparticles. The purified nanoparticles are efficiently bound to the surface of UT7/Epo-S1 cells, which are semi-permissive hosts for B19V infection. Immunization of BALB/c mice with VP1u 5-80 nanoparticles elicited a robust production of B19V-specific IgG antibodies compared to single VP1u 5-80 peptides. Moreover, a neutralization assay using B19V derived from a blood donor sample revealed that antibodies from mice immunized with VP1u 5-80 nanoparticles exhibited stronger infection-neutralizing activity. These findings suggest that nanoparticle formation plays a crucial role in enhancing the immunogenicity of the B19V VP1u 5-80 amino acid peptide and that these nanoparticles could serve as promising vaccine candidates, effectively inducing immunity against B19V.

EPCO-53. CHARACTERIZING THE FUNCTIONAL IMPACT OF GERMLINE-INHERITED RISK ALLELES IN GLIOMA EVOLUTION

Abstract Efforts to understand etiology of glioma led researchers to investigate associated genetic factors. Genome Wide Association Studies (GWAS) conducted to that end, uncovered an association of various single nucleotide polymorphisms (SNP) among hallmark oncogenes and tumor suppressors as risk variants in glioma incidence. Here, we perform in vitro modeling of four risk alleles/SNPs((rs723527 (EGFR), rs55705857 (MYC), rs7572263 (IDH1), rs78378222 (TP53)) mapped to non-coding regulatory regions of the genome, to assess their causal contribution to glioma evolution. To model the aforementioned risk alleles in an in-vitro system, we used a CRISPR-knock in strategy in human induced pluripotent stem cells (hiPSCs) and verified by Sanger sequencing. To study the effects of the risk alleles on brain cell types, we generated a cerebral organoid model from the edited and control lines and subjected them to downstream morphological and transcriptomic analyses. While we did not see morphological changes in the histological sections relative to controls at the early 14-day timepoint, we observed increased differentiation in both control and risk cohorts, along with an absence of neural rosettes, at the late 90-day timepoint. Bulk RNA-seq of the organoids at 14-days resulted in positive enrichment of oncogenic signaling pathways and negative enrichment of DNA repair pathways in the risk allele cohorts. Furthermore, single cell RNA seq of 14 and 90-day samples revealed a sharp decrease in neural stem cell clusters in both IDH and TP53 cohorts, while control organoids demonstrated robust production of immature neurons at 90 days. Overall, we observed a significant increase in differentiation into mature cell clusters in the control, relative to the risk allele organoids. Ongoing efforts are aimed at understanding this apparent differentiation block in the IDH and TP53 compartments, relative to control. Further analysis will lead to addressing key understudied areas of glioma etiology and associated mechanisms of risk incidence.

Fast and robust production of quantum superposition states by the fractional shortcut to adiabaticity

The fractional shortcut to adiabaticity (f-STA) for the production of quantum superposition states is proposed firstly via a three-level system with a Λ-type linkage pattern and a four-level system with a tripod structure. The fast and robust production of the coherent superposition states is studied by comparing the populations for the f-STA and the fractional stimulated Raman adiabatic passage (f-STIRAP). The states with equal proportions can be produced by fixing the controllable parameters of the driving pulses at the final moment of the whole process. The effects of the pulse intensity and the time delay of the pulses on the production process are discussed by monitoring the populations on all of the quantum states. In particular, the spontaneous emission arising from the intermediate state is investigated by the quantum master equation. The result reveals that the f-STA exhibits superior advantages over the f-STIRAP in producing the superposition states.

Muramyl Dipeptide-Presenting Polymersomes as Artificial Nanobacteria to Boost Systemic Antitumor Immunity.

The clinical efficacy of cancer vaccines is closely related to immunoadjuvants that play a crucial role in magnifying and prolonging the immune response. Muramyl dipeptide (MDP), a minimal and conserved peptidoglycan found in almost all bacteria, can trigger robust immune activation by uniquely antagonizing the nucleotide-binding oligomerization domain 2 (NOD2) pathway. However, its effectiveness has been hindered by limited solubility, poor membrane penetration, and rapid clearance from the body. Here, we introduce MDP-presenting polymersomes as artificial nanobacteria (NBA) to boost the antitumor immune response. The NBA, featuring abundant MDP molecules, induces superior stimulation of immune cells including macrophages and bone marrow-derived dendritic cells (BMDCs) compared to free MDP, likely via facilitating immune cell uptake and cooperatively stimulating systemic NOD2 signaling. Importantly, systemic administration of NBA significantly enhances the chemo-immunotherapy of B16-F10 melanoma-bearing mice pretreated with doxorubicin by reversing the immunosuppressive tumor microenvironment. Furthermore, NBA carrying ovalbumin and B16-F10 cell lysates induces robust OVA-IgG antibody production and effectively inhibit tumor growth, respectively. The artificial nanobacteria hold great promise as a potent systemic immunoadjuvant for cancer immunotherapy.

Advancing biopharmaceutical manufacturing: economic and sustainability assessment of end-to-end continuous production of monoclonal antibodies

Monoclonal antibodies (mAbs) have become essential therapeutics for treating various diseases. The robust, cost-effective, and sustainable production of mAbs is crucial due to their growing clinical and commercial demand. Advances in bioprocessing, such as improved cell lines, perfusion bioreactors, multicolumn chromatography, and automation, can significantly increase productivity, making treatments more accessible. Streamlining the production process also aligns with environmental sustainability by reducing waste and energy consumption. This study quantifies the economic and environmental impacts of incorporating recent advances into end-to-end continuous bioprocessing of mAbs. The results demonstrate that, compared with an optimized best-in-class fed-batch process (with 15 g/l titer and multicolumn chromatography), continuous manufacturing can reduce the total annual production costs, facility footprint, plastic waste, and CO2 emissions by up to 23%, 51%, 57%, and 54%, respectively, in a producing clinical and commercial lots. Additionally, uncertainty analysis indicates that these gains are even more substantial under demand fluctuations.

Enhanced charge transfer kinetics across the graphdiyne-metal sulfide ohmic interface for boosting photocatalytic hydrogen evolution

The design and construction of photocatalysts are the crucial step for solar-driven hydrogen evolution. Herein, a new type of graphdiyne (CnH2n-2)-based photocatalyst, which was prepared by one pot method, was used to promote hydrogen evolution reaction by constructing GDY/VS2 ohmic junction with S sites anchoring on the surface. Actually, the unique two-dimensional conjugated carbon network of GDY can maintain the structural stability. The uneven surface facilitates the flow of electrons, which makes GDY a good electron donor. In addition, the expanded interlayer spacing of VS2 exposes more hydrogen evolution active sites. The increase in surface electronegativity of composite catalysts enhances their redox ability and charge transfer kinetics. The band edge of the graphdiyne-metal sulfide Ohmic contact interface is bent, allowing electrons to flow into the co catalyst without resistance. These synergistic effects lead to the robust hydrogen production of GDY/VS2, which was increased by nearly 10.3 times compared with that of pure GDY. Finally, charge migration at the interface of graphdiyne-metal sulfide heterojunction was proposed and further verified combining in situ XPS and theory calculation. This research marks a significant stride for enhance the efficiency of photocatalytic hydrogen evolution by designing and constructing composite heterojunction materials.

Multi-Epitopic Peptide Vaccine Against Newcastle Disease Virus: Molecular Dynamics Simulation and Experimental Validation

Background: Newcastle disease virus (NDV) is a highly contagious and economically devastating pathogen affecting poultry worldwide, leading to significant losses in the poultry industry. Despite existing vaccines, outbreaks continue to occur, highlighting the need for more effective vaccination strategies. Developing a multi-epitopic peptide vaccine offers a promising approach to enhance protection against NDV. Objectives: Here, we aimed to design and evaluate a multi-epitopic vaccine against NDV using molecular dynamics (MD) simulation. Methodology: We retrieved NDV sequences for the fusion (F) protein and hemagglutinin–neuraminidase (HN) protein. Subsequently, B-cell and T-cell epitopes were predicted. The top potential epitopes were utilized to design the vaccine construct, which was subsequently docked against chicken TLR4 and MHC1 receptors to assess the immunological response. The resulting docked complex underwent a 1 microsecond (1000 ns) MD simulation. For experimental evaluation, the vaccine’s efficacy was assessed in mice and chickens using a controlled study design, where animals were randomly divided into groups receiving either a local ND vaccine or the peptide vaccine or a control treatment. Results: The 40 amino acid peptide vaccine demonstrated strong binding affinity and stability within the TLR4 and MHC1 receptor–peptide complexes. The root mean square deviation of peptide vaccine and TLR4 receptor showed rapid stabilization after an initial repositioning. The root mean square fluctuation revealed relatively low fluctuations (below 3 Å) for the TLR4 receptor, while the peptide exhibited higher fluctuations. The overall binding energy of the peptide vaccine with TLR4 and MHC1 receptors amounted to −15.7 kcal·mol−1 and −36.8 kcal·mol−1, respectively. For experimental evaluations in mice and chicken, the peptide vaccine was synthesized using services of GeneScript Biotech® (Singapore) PTE Limited. Experimental evaluations showed a significant immune response in both mice and chickens, with the vaccine eliciting robust antibody production, as evidenced by increasing HI titers over time. Statistical analysis was performed using an independent t-test with Type-II error to compare the groups, calculating the p-values to determine the significance of the immune response between different groups. Conclusions: Multi-epitopic peptide vaccine has demonstrated a good immunological response in natural hosts.

Robust methane production from anaerobic digestion of maize silage: Feeding control using sliding mode control strategy with Anaerobic Digestion Model N°01

This paper introduces a robust control approach based on the Sliding Mode Control (SMC) strategy for the AD process. We use the Anaerobic Digestion Model N°01 (ADM1), the most detailed and comprehensive model for AD, as a virtual AD plant. The proposed control scheme is derived from a reduced model. This control law uses the state variables of the reduced AM2HN model as well as the stoichiometric and kinetic parameters. An interface block between the ADM1 and AM2HN state variables has been designed in a closed loop. The performance of SMC has been enhanced by the use of a low-pass filter, which completely eliminates chattering and provides smooth dilution rates (chatter-free) while rapidly reaching the target methane production (in less than 5 days) in a noisy environment.

Metabolic activation of WHO-congeners PCB28, 52, and 101 by human CYP2A6: evidence from in vitro and in vivo experiments.

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4'-trichlorobiphenyl (PCB28), 2,2',5,5'-tetrachlorobiphenyl (PCB52), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61µg/L (5,89nM) for PCB28, 316.98µg/L (1,03µM) for PCB52, and 151.1µg/L (441nM) for PCB101 from a 20µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners.

Efficient 7-Dehydrocholesterol Production by Multiple Metabolic Engineering of Diploid Saccharomyces cerevisiae.

7-Dehydrocholesterol (7-DHC), a direct precursor of vitamin D3, has attracted increasing attention in microbial fermentation recently. In this study, 7-DHC biosynthesis in diploid Saccharomyces cerevisiae with robust ergosterol production was achieved by heterologous 24-dehydrocholesterol reductase expression, generating 44.1 mg/L 7-DHC, whereas the titer of ergosterol decreased by 40.5%. The ergosterol biosynthetic pathway was completely blocked by knocking out ERG6 and ERG5, affording a 4.2-fold increase in the 7-DHC titer. Subsequently, the facilitation of the mevalonate and the postsqualene pathways accompanied by elimination of transcriptional repressors enhanced 7-DHC synthesis, and the 7-DHC titer reached 738.5 mg/L in a shake flask. Further validation in a 50 L fermenter demonstrated that the 7-DHC titer reached 3.80 g/L within just 24 h, with productivity reaching 158.3 mg/L/h, setting a new benchmark as the highest reported to date. This study paves the way toward a large-scale and cost-effective manufacture of 7-DHC.

Predicting the Microbiome and Metabolome Dynamics of Natural Apple Fermentation Towards the Development of Enhanced Functional Vinegar

Natural vinegar fermentation is a complex process influenced by the interplay between microbial communities and metabolites. This study examined the interplay between the microbiome and the metabolome over a three-month period, with samples collected every ten days. Using Illumina sequencing and chromatographic techniques (HPLC and GC-MS), we mapped microbial shifts and metabolite profiles. Early fermentation showed a diverse microbial presence, including genera such as Cronobacter, Luteibacter, and Saccharomyces. A stable microbial ecosystem established between days 15 and 70, characterized by the dominance of Leuconostoc, Gluconobacter, and Saccharomyces, which facilitated consistent substrate consumption and metabolite production, including various organic acids and ethanol. By day 70, Acetobacter prevalence increased significantly, correlating with a peak acetic acid production of 12.4 g/L. Correlation analyses revealed significant relationships between specific microbes and volatile organic compounds. This study highlights the crucial roles of these microbes in developing sensory profiles suited for industrial applications and proposes an optimal microbial consortium for enhancing vinegar quality. These data suggest that an optimal microbial consortium for vinegar fermentation should include Saccharomyces for efficient alcohol production, Leuconostoc for ester-mediated flavor complexity, and Acetobacter for robust acetic acid production. The presence of Komagataeibacter could further improve the sensory and functional qualities due to its role in producing bacterial cellulose.

NIR-Guided Coating Optimization of Omega-3 Fatty Acid Mini Soft Capsules with Pitavastatin and Ezetimibe

Background: This study aimed to optimize the coating process of Omega-3 fatty acid (OM3-FA) mini soft capsules containing the active pharmaceutical ingredients (APIs) pitavastatin and ezetimibe using near-infrared (NIR) spectroscopy for in-process monitoring. Cardiovascular disease treatments benefit from combining OM3-FA with lipid-lowering agents, but formulating such combinations in mini soft capsules presents challenges in maintaining stability and mechanical integrity. Methods: The coating process was developed using a pan coater and real-time NIR monitoring to ensure uniformity and quality. NIR spectroscopy enabled precise control of coating thickness, ensuring consistent drug distribution across the capsule surface. Results: The optimized process minimized OM3-FA oxidation and preserved the mechanical integrity of the capsules, as confirmed by texture analysis and in-vitro dissolution testing. This integration of NIR spectroscopy as a process analytical technology (PAT) significantly improved coating quality control, resulting in a stable and effective combination therapy for pitavastatin and ezetimibe in a mini soft capsule form. Conclusion: This approach offers an efficient solution for enhancing patient adherence in cardiovascular disease management. The application of NIR spectroscopy for real-time monitoring highlights its broader significance in pharmaceutical manufacturing, where it can serve as a versatile tool for ensuring product quality and optimizing production efficiency in diverse formulation processes. By incorporating NIR-based PAT, manufacturers can not only achieve product-specific improvements but also establish a foundation for continuous manufacturing and automated quality assurance systems, ultimately contributing to a more streamlined and robust production environment.

Dietary Probiotic Pediococcus acidilactici GKA4, Dead Probiotic GKA4, and Postbiotic GKA4 Improves Cisplatin-Induced AKI by Autophagy and Endoplasmic Reticulum Stress and Organic Ion Transporters.

Acute kidney injury (AKI) syndrome is distinguished by a quick decline in renal excretory capacity and usually diagnosed by the presence of elevated nitrogen metabolism end products and/or diminished urine output. AKI frequently occurs in hospital patients, and there are no existing specific treatments available to diminish its occurrence or expedite recovery. For an extended period in the food industry, Pediococcus acidilactici has been distinguished by its robust bacteriocin production, effectively inhibiting pathogen growth during fermentation and storage. In this study, the aim is to assess the effectiveness of P. acidilactici GKA4, dead probiotic GKA4, and postbiotic GKA4 against cisplatin-induced AKI in an animal model. The experimental protocol involves a ten-day oral administration of GKA4, dead probiotic GKA4, and postbiotic GKA4 to mice, with a cisplatin intraperitoneal injection being given on the seventh day to induce AKI. The findings indicated the significant alleviation of the renal histopathological changes and serum biomarkers of GKA4, dead probiotic GKA4, and postbiotic GKA4 in cisplatin-induced nephrotoxicity. GKA4, dead probiotic GKA4, and postbiotic GKA4 elevated the expression levels of HO-1 and decreased the expression levels of Nrf-2 proteins. In addition, the administration of GKA4, dead probiotic GKA4, and postbiotic GKA4 significantly reduced the expression of apoptosis-related proteins (Bax, Bcl-2, and caspase 3), autophagy-related proteins (LC3B, p62, and Beclin1), and endoplasmic reticulum (ER) stress-related proteins (GRP78, PERK, ATF-6, IRE1, CHOP, and Caspase 12) in kidney tissues. Notably, GKA4, dead probiotic GKA4, and postbiotic GKA4 also upregulated the levels of proteins related to organic anion transporters and organic cation transporters. Overall, the potential therapeutic benefits of GKA4, dead probiotic GKA4, and postbiotic GKA4 are significant, particularly after cisplatin treatment. This is achieved by modulating apoptosis, autophagy, ER stress, and transporter proteins to alleviate oxidative stress.

Simulation-Based Optimization Workflow of CO2-EOR for Hydraulic Fractured Wells in Wolfcamp A Formation

Hydraulic fracturing has enabled production from unconventional reservoirs in the U.S., but production rates often decline sharply, limiting recovery factors to under 10%. This study proposes an optimization workflow for the CO2 huff-n-puff process for multistage-fractured horizontal wells in the Wolfcamp A formation in the Delaware Basin. The potential for enhanced oil recovery and CO2 sequestration simultaneously was addressed using a coupled geomechanics–reservoir simulation. Geomechanical properties were derived from a 1D mechanical earth model and integrated into reservoir simulation to replicate hydraulic fracture geometries. The fracture model was validated using a robust production history matching. A fluid phase behavior analysis refined the equation of state, and 1D slim tube simulations determined a minimum miscibility pressure of 4300 psi for CO2 injection. After the primary production phase, various CO2 injection rates were tested from 1 to 25 MMSCFD/well, resulting in incremental oil recovery ranging from 6.3% to 69.3%. Different injection, soaking and production cycles were analyzed to determine the ideal operating condition. The optimal scenario improved cumulative oil recovery by 68.8% while keeping the highest CO2 storage efficiency. The simulation approach proposed by this study provides a comprehensive and systematic workflow for evaluating and optimizing CO2 huff-n-puff in hydraulically fractured wells, enhancing the recovery factor of unconventional reservoirs.

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity.

Tumor progression is associated with tumor-cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte-mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host-guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte-mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

A Mechanical Immune Checkpoint Inhibitor Stiffens Tumor Cells to Potentiate Antitumor Immunity

Tumor progression is associated with tumor‐cell softening. Improving the stiffness of the tumor cells can make them more vulnerable to lymphocyte‐mediated attack. Tumor cell membranes typically exhibit higher cholesterol levels than normal cells, making tumor cells soft. Herein, we demonstrate a mechanical immune checkpoint inhibitor (MICI) formulated by cyclodextrin (CD) lipids and fusogenic lipids. Through fusing CD lipids into the tumor cell membrane using a fusogenic liposome formulation, the cholesterol in the plasma membrane is reduced due to the specific host–guest interactions between CD lipid and cholesterol. As a result, tumor cells are stiffened, and the activation of lymphocytes (including NK and cytotoxic effector T cells) is improved when contacting the stiffened tumor cells, characterized by robust degranulation and effector cytokine production. Notably, this treatment has negligible influence on the infiltration and proliferation of lymphocytes in tumor tissues, confirming that the enhanced antitumor efficacy should result from activating a specific number of lymphocytes caused by direct regulation of the tumor cell stiffness. The combination of MICIs and clinical immunotherapies enhances the lymphocyte‐mediated antitumor effects in two tumor mouse models, including breast cancer and melanoma. Our research also reveals an unappreciated mechanical dimension to lymphocyte activation.

An Adhesive Hydrogel Technology for Enhanced Cartilage Repair: A Preliminary Proof of Concept.

Knee cartilage has limited natural healing capacity, complicating the development of effective treatment plans. Current non-cell-based therapies (e.g., microfracture) result in poor repair cartilage mechanical properties, low durability, and suboptimal tissue integration. Advanced treatments, such as autologous chondrocyte implantation, face challenges including cell leakage and inhomogeneous distribution. Successful cell therapy relies on prolonged retention of therapeutic biologicals at the implantation site, yet the optimal integration of implanted material into the surrounding healthy tissue remains an unmet need. This study evaluated the effectiveness of a newly developed photo-curable adhesive hydrogel for cartilage repair, focusing on adhesion properties, integration performance, and ability to support tissue regeneration. The proposed hydrogel design exhibited significant adhesion strength, outperforming commercial adhesives such as fibrin-based glues. An in vivo goat model was used to evaluate the hydrogels' adhesion properties and long-term integration into full-thickness cartilage defects over six months. Results showed that cell-free hydrogel-treated defects achieved superior integration with surrounding tissue and enhanced cartilage repair, with notable lateral integration. In vitro results further demonstrated high cell viability, robust matrix production, and successful cell encapsulation within the hydrogel matrix. These findings highlight the potential of adhesive hydrogel formulations to improve the efficacy of cell-based therapies, offering a potentially superior treatment for knee cartilage defects.

B-086 Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure

Abstract Background The immune response to SARS-CoV-2 becomes increasingly complex as individuals receive different combinations of vaccines and encounter breakthrough infections. Our study aims to describe the immunogenicity observed over a two-year period in healthy individuals who completed two-dose series and then experienced booster and/or Omicron infection. Methods In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center. At 1, 5, 11, and 25 months after second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses using commercially available immunoassays and surrogate virus neutralization tests, along with interferon gamma (IFN-γ)-based enzyme-linked immunosorbent spot (ELIspot) assay using SARS-CoV-2 spike peptide pools. Results As the humoral immune response was monitored longitudinally, robust antibody production persisted after the third antigen exposure, in contrast to the rapid decline observed between the second and third dose injections. All immune metrics tended to increase as the number of vaccine doses increased. We also found a trend toward increased humoral immune response in the homologous mRNA-based vaccine group and enhanced cellular immune response in the vector-based primary series with mRNA booster heterologous combination group. Interestingly, no significant difference was found between Omicron exposure through breakthrough infection and bivalent vaccination. Omicron exposure significantly increased the breadth of neutralizing activity, while it did not significantly alter the cross-reactivity in the cellular immune response. Conclusions We identified individuals with diminished neutralization, aiding decisions for future vaccination prioritization. In addition, our findings suggest the role of cellular immunity as an important correlate of long-term protection through cross-reactivity to emerging variants.

Effect of bacterial fermentation on the ability of myofibrillar proteins to bind esters and its potential mechanism: Based on protein metabolism and structural changes

The effect of fermentation strains (Lactiplantibacillus plantarum CQ01107 and Staphylococcus simulans CD207) on the binding properties of porcine myofibrillar proteins (MPs) to esters was investigated from two perspectives: metabolism degree and structural alterations. Results demonstrated that S. simulans could reduce the particle size and α-helix content of MPs, while simultaneously increasing the absolute zeta potential and active sulfhydryl content. This process decreased protein aggregation and facilitated the unfolding of MPs, thereby enhancing their binding to esters. Conversely, L. plantarum showed limited promotion, which might be related to its robust acid production and protein hydrolysis capacities. In addition, ethyl octanoate, with a longer carbon chain length, was found to have the highest binding capacity to MPs (28.38 %–41.59 %). Molecular docking results further revealed that the binding of the four esters to MPs was spontaneous, with ethyl octanoate exhibiting the lowest binding energy to MPs (−5.635 kcal/mol). The primary forces involved in the binding of the four selected esters to MPs were hydrophobic interactions, hydrogen bonding, and van der Waals forces. These findings can provide new insights into the mechanisms by which fermentation strains influence flavor formation in fermented foods.