Search Results (754)

Phosphatidylserine (PS), a valuable phospholipid, is widely used in food, pharmaceutical and cosmetic industries. Its enzymatic synthesis, catalyzed by phospholipase D (PLD) via transphosphatidylation under mild conditions, has drawn considerable attention. However, the industrial use of free PLD is limited by poor stability, difficult recovery, and high cost. To address these challenges, a ternary composite carrier—integrating the flexibility of chitosan, the stability of cellulose, and the macroporosity of agarose—was constructed for immobilizing the PLD from Streptomyces antibioticus (saPLD). The resulting saPLD@chitosan–cellulose–agarose biocatalyst demonstrated enhanced immobilization efficiency, catalytic performance, and stability across varying pH and temperatures. After eight consecutive batches of usage, the PS yield of saPLD@chitosan–cellulose–agarose reached over 60% of that from the first batch. Thus, this study established a new method for preparing immobilized saPLD, and developed a robust and promising platform for the efficient and sustainable production of PS. Full article

The design and optimization of immobilized metal affinity chromatography (IMAC) media are crucial to enhancing the purification efficiency of recombinant proteins. In this study, the agarose-based microspheres are prepared by using a three-factorial Box–Behnken design followed by NTA-Ni²⁺ agarose-based microspheres (ABM) preparation by the “one-step” crosslinking of epichlorohydrin (ECH)–nitrilotriacetic acid (NTA) to efficiently couple the NTA ligand to the surface of the matrix. After preparation, various sophisticated techniques, including SEM, AFM, DSC, FTIR, and SDS-PAGE, were used to analyze the morphological structure, thermal stability, and chemical composition of NTA-Ni²⁺ ABM. The optimal conditions are identified as an emulsifier PP concentration of 8.12 wt%, a stirring speed of 1624.46 rpm, and an oil-phase temperature of 53.86 °C, giving a span value (Y) of 0.50684. SEM, AFM, DSC, and FTIR results showed that the fabricated NTA-Ni²⁺ ABM were structurally stable and had a uniform cross-linking network for up to 8 h of coupling reaction time. The performance results showed that the beads had a high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL), and SDS-PAGE results demonstrated the efficient purification ability for target proteins. These findings provide the theoretical basis and a practical solution for the rational design and application of IMAC medium. Full article

Biodegradable hydrogel-based conductive inks, with application in additive circuit manufacturing, are synthesized from agarose, sodium alginate and functional carbon-based particles (carbon nanotubes and graphite). Rheological measurements are conducted to evaluate the printing performance of each ink. The synthesized functional inks are printed, and their conductivity properties are evaluated as a function of the functional material concentration. Promising conductivity values are achieved, demonstrating their potential application for low-cost and low-environmental-impact circuital and electromagnetic designs. Full article

The lipase B from Candida antarctica was immobilized on octyl-agarose using low and high (one that saturated the support surface with enzyme) loadings. Then, both biocatalysts were aminated, and the aminated and non-aminated biocatalysts were used in further experiments. The enzyme activity was determined using substrates with different structures. The modification of the four biocatalysts with poly (ethylene-alt-maleic anhydride) revealed that only a marginal covalent reaction occurs. That way, the ion exchange of the polymer on the immobilized enzyme surface should be responsible for the enzyme functional changes. The modification of the biocatalysts with this polymer produced mixed results for enzyme activity (depending on the enzyme loading, use of aminated or non-aminated enzyme, polymer concentration and used substrate), in some instances more than doubling the activity, in others reducing it by 5–6 times the activity when compared to the unmodified biocatalyst. The effects on biocatalyst stability were also mixed, depending on the same factors; in some instances, great stabilization could be found (e.g., in inactivation of the highly loaded aminated biocatalyst at pH 7.0, the unmodified biocatalyst kept 5% of the initial activity, while the biocatalyst modified with 1% of the polymer maintained 80%), but in other instances, enzyme stability was reduced after modification. It was shown that one of the effects of the polymer modification was the prevention of the enzyme release during inactivation. Full article

Culturing cells and micro-tissue samples in 3D bio-scaffolding structures is gaining popularity; however, precise control of tissue micro-environment in such systems remains challenging. We describe a family of new hybrid bio-scaffolds with 3D O₂-sensing ability, produced by simple means from readily available bio-scaffolding and O₂-sensing materials. Three different types of phosphorescent O₂-sensing materials—polymeric microparticles (MPs), supramolecular probe MitoXpress and nanoparticulate probes NanO2 and Nano-IR (NPs)—were integrated in Matrigel and agarose scaffolding materials and evaluated. Key working characteristics of such hybrid scaffolds, including heterogeneity, stability, cytotoxicity, optical signals and O₂-sensing properties, ease of fabrication and use, were compared. The results show superiority of the Matrigel hybrids with NanO2 and Nano-IR probes. Demonstration experiments were conducted with HCT116 cells and individual spheroids derived from these cells, culturing them in the Matrigel–NP hybrid scaffolds and monitoring oxygenation and local O₂ gradients on a time-resolved fluorescence plate reader and by phosphorescence lifetime imaging microscopy (PLIM). Full article

The fungal pathogen Metschnikowia bicuspidata causes “milky disease” in the Chinese mitten crab (Eriocheir sinensis), which poses substantial challenges to sustainable aquaculture development considering the current lack of effective treatment interventions. To address this issue, in laboratory validation, we developed two rapid recombinase polymerase amplification (RPA) detection methods for M. bicuspidata in E. sinensis targeting the histone acetyltransferase B-type subunit 2 gene (HAT-B2): an electrophoretic assay (RPA-AGE) and a colloidal gold lateral flow dipstick assay (RPA-LFD). We optimized RPA-AGE and RPA-LFD protocols for specific pathogen detection. Target detection was achieved within 35 min using RPA-AGE (30 min amplification at 37 °C followed by 5 min agarose gel electrophoresis), whereas RPA-LFD provided results in 15 min with high specificity (10 min amplification at 37 °C plus 5 min strip reading). Both methods exhibited exclusive specificity to M. bicuspidata, with no cross-reactivity with six pathogens, including Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Candida albicans, Saccharomyces cerevisiae, and Microsporidia sp. The detection sensitivity of both platforms reached 4.8 copies/μL in laboratory validation. For field testing, the detection results from 30 field samples showed that although the 70% detection rate was lower than the 83.3% achieved by quantitative PCR, these approaches surpassed the detection rate of conventional PCR (53.3%). Notably, the RPA-LFD platform is applicable under field conditions as no specialized equipment is required. These rapid, sensitive, and specific detection systems provide practical tools for the early diagnosis and containment of M. bicuspidata infections in aquaculture settings. Full article

Accurate genotyping of small insertions and deletions (InDels; <5 bp) remains technically challenging in routine molecular breeding, largely due to the limited resolution of agarose gel electrophoresis and the labor-intensive nature of polyacrylamide-based assays. Here, we present the Tri-Primer Amplification Refractory Mutation System (TP-ARMS), a simple and cost-effective PCR-based strategy that enables high-resolution genotyping of small InDels using standard agarose gels. The TP-ARMS employs a universal reverse primer in combination with two allele-specific forward primers targeting insertion and deletion alleles, respectively. This design allows clear discrimination of homozygous and heterozygous genotypes using a two-tube PCR workflow. The method showed complete concordance with Sanger sequencing in detecting 1–5 bp InDels across multiple crop species, including rice (Oryza sativa) and quinoa (Chenopodium quinoa). In addition, using a TP-ARMS reduced experimental time by approximately 90% compared with PAGE-based approaches and avoided the high equipment and DNA quality requirements of fluorescence-based assays. The practical applicability of the TP-ARMS was demonstrated in breeding populations, including efficient genotyping of a 3-bp InDel in OsNRAMP5 associated with cadmium accumulation and a 6-bp promoter InDel in OsSPL10 underlying natural variation in rice trichome density across 370 accessions. Collectively, the TP-ARMS provides a robust, scalable, and low-cost solution for precise small InDel genotyping, with broad applicability in marker-assisted breeding and functional genetic studies. Full article

Plasma cell disorders often have urinary excretion of monoclonal immunoglobulins and renal injury that are evaluated using total protein assays and electrophoresis. Proteinuria was evaluated for 100 patients with plasma cell disorders and 24 h collections. A turbidimetric method on the Abbott Alinity quantified protein. Electrophoresis used agarose gels. Most specimens (66%) had protein concentrations below the manufacturer’s limit of quantitation (LOQ), 6.8 mg/dL (68 mg/L). After validating an LOQ of 3 mg/dL (30 mg/L), 34% of urine specimens still were below the LOQ. After excluding 40 patients with other causes of increased protein excretion (decreased estimated glomerular filtration rate (eGFR), diabetes mellitus, or overflow proteinuria), almost all patients had protein excretion below an upper reference limit of 150 mg/d. Median total protein excretion for these 60 patients was 75 mg/d; only one patient excreted >132 mg/d. However, 32% of these patients without increased total protein excretion had albumin excretion ≥ 30 mg/d, suggestive of kidney injury. Electrophoretic patterns included glomerular, tubular, and overflow proteinuria; 32 specimens contained monoclonal immunoglobulins. Protein concentrations of urine are often below LOQs of total protein assays, raising questions about whether LOQs should be improved. Urine albumin measurements and electrophoretic patterns may serve as more sensitive indicators of kidney injury in patients with plasma cell disorder than measurements of total protein excretion or increased serum creatinine. Full article

The flowers of Panax ginseng C. A. Mey. (PG) and Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow (PN) are morphologically indistinguishable after drying, leading to prevalent adulteration that compromises product quality and consumer safety. To address this issue, we developed a rapid, closed-tube molecular authentication method based on high-resolution melting (HRM) analysis. Species-specific primer pairs were designed to target the conserved ITS and rbcL-accD regions, with PNG-2 selected as the optimal candidate owing to its stable genotyping performance and moderate GC content. Our results established GC content, rather than amplicon length, as the primary determinant of the melting temperature (Tm). Notably, the experimentally measured Tm values were consistently 0.7–1.5 °C higher than theoretical predictions, a discrepancy attributable to the stabilizing effect of the saturated fluorescent dye. To ensure maximum diagnostic reliability, the HRM results were cross-validated through a three-tier system comprising ITS2 phylogenetic analysis, agarose gel electrophoresis, and Sanger sequencing. The practical utility and matrix robustness of the assay were further verified using a diversified validation cohort of 30 commercial samples, including 24 floral batches and 6 root-derived products (root slices and ultramicro powders). The HRM profiles demonstrated 100% concordance with DNA barcoding results, effectively identifying mislabeled products across different botanical matrices and processing forms. This methodology, which can be completed within 3 h, provides a significantly more cost-effective and rapid alternative to traditional sequencing-based methods for large-scale market surveillance and industrial quality control. Full article

Cleaning historical silk textiles is a particularly sensitive operation that requires precise control to prevent mechanical or chemical damage. In this study, we investigate using flexible PVA–borax-based gels to remove soot from silk, i.e., polyvinyl alcohol–borax (PVA-B) gels and polyvinyl alcohol–borax–agarose double network gels (PVA-B/AG DN) loaded with different cleaning agents—namely, 30% ethanol and 1% Ecosurf EH-6—in addition to plain gels loaded with water. These gel formulations were tested on simplified model systems (SMS) and were applied using two methods: placing and tamping. The cleaning results were compared with a traditional contact-cleaning approach; micro-vacuuming followed by sponging. Visual inspection, 3D opto-digital microscopy, colorimetry, and machine-learning-assisted (ML) soot counting were exploited for the assessment of cleaning efficacy. Rheological characterization provided information about the flexibility and handling properties of the different gel formulations. Among the tested systems, the DN gel containing only water, applied by tamping, was easy to handle and demonstrated the highest soot-removal effectiveness without leaving residues, as confirmed by micro-Fourier Transform Infrared (micro-FTIR) analysis. Scanning electron microscope (SEM) micrographs proved the structural integrity of the treated silk fibers. Overall, this work allows us to conclude that PVA–borax-based gels offer an effective, adaptable, and low-risk cleaning strategy for historical silk fabrics. Full article

Needle-free injection (NFI) technology is a promising alternative to conventional syringe injection, as it mitigates needle-related complications and enhances patient compliance. However, achieving the controlled and efficient dispersion of larger-volume formulations (>1 mL) within tissues remains a significant challenge. This study presents a novel pneumatic NFI system that uses a two-phase driving mode to regulate driving pressure and duration with an ejection volume of 1.0–2.0 mL. The integrated pressure stabilization unit significantly reduces pressure fluctuations during the initial injection phase, generating a more stable and uniform spray distribution. It is designed to produce an ideal elliptical dispersion effect while eliminating splatter, enabling controlled large-volume delivery. Jet impact experiments were conducted to investigate the dynamic characteristics of microjets generated by conventional single-phase and novel two-phase driving modes. Furthermore, the influence of the driving mode on the dispersion behaviors of microjets in agarose gels was explored through high-speed imaging of gel injections. The results demonstrate that the two-phase driving mode produces a distinct two-phase jet pressure profile. Compared to the single-phase mode, the two-phase mode produced a significantly larger dispersion width at equivalent initial driving pressures. This promotes more uniform lateral drug distribution and achieves a higher percentage of liquid drug delivery in gels. Furthermore, favorable driving pressure combinations were identified for different volumes: (1.25–0.25) MPa for 1.0 mL, (1.25–0.50) MPa for 1.5 mL, and (1.50–0.50) MPa for 2.0 mL. This provides a practical basis for optimizing clinical parameters and advancing the development of controllable NFI systems. Full article

Chymosin is an aspartyl protease widely used in the food industry for milk coagulation during cheesemaking. Although recombinant production has replaced natural extraction from rennet, current heterologous expression systems still face significant challenges, including low solubility, costly purification steps, and enzyme instability after activation. To address these limitations, we sought to develop a more efficient and economical production strategy for bovine chymosin by cloning its codon-optimized prochymosin A gene into Escherichia coli using the pBAD/His vector under the control of the L-arabinose-inducible PBAD promoter. Overexpression of the recombinant gene resulted in the formation of inclusion bodies, which were solubilized with NaOH and refolded by dilution and pH adjustment with glycine. The folded prochymosin was then activated by acidification. To simplify the downstream process and improve enzyme recovery, different immobilization strategies were explored to combine activation, purification, and immobilization in a single step. While polymeric agarose-based supports showed low immobilization efficiency (<20%) due to pore clogging, magnetic nanoparticles completely overcame these limitations, achieving nearly 100% immobilization yield and retaining about 85% of enzymatic activity. This integrated magnetic-based approach provides a cost-effective and scalable alternative for the production and stabilization of active chymosin. Full article

The combination of gold nanoparticles (AuNPs) with hydrogels has drawn significant interest in the design of smart materials as advanced platforms for biomedical applications. These systems endow light-responsiveness enabled by the AuNPs localized surface plasmon resonance (LSPR) phenomenon. In this study, we propose a nanocomposite hydrogel in which gold nanorods (AuNRs) are included in an agarose–carbomer–hyaluronic acid (AC-HA)-based hydrogel matrix to study the correlation between light irradiation, local temperature increase, and drug release for potential light-assisted drug delivery applications. The gel is obtained through a facile microwave-assisted polycondensation reaction, and its properties are investigated as a function of both the hyaluronic acid molecular weight and ratio. Afterwards, AuNRs are incorporated in the AC-HA formulation, before the sol–gel transition, to impart light-responsiveness and optical properties to the otherwise inert polymeric matrix. Particular attention is given to the evaluation of AuNRs/AC-HA light-induced heat generation and drug delivery performances under near-infrared (NIR) laser irradiation in vitro. Spatiotemporal thermal profiles and high-resolution thermal maps are registered using fiber Bragg grating (FBG) sensor arrays, enabling accurate probing of maximum internal temperature variations within the composite matrix. Lastly, using a high-steric-hindrance protein (BSA) as a drug mimetic, we demonstrate that moderate localized heating under short-time repeated NIR exposure enhances the release from the nanocomposite hydrogel. Full article

Background: Survivin is an anti-apoptotic protein highly expressed during embryonic development and, in adults, mainly in the gastrointestinal epithelium. Its levels decrease in human gastric tissue and cultured cells upon exposure to Helicobacter pylori gamma-glutamyl transpeptidase (GGT), though the underlying mechanism remains unclear. Objective: We aimed to investigate the role of cap-independent translation driven by the Survivin 5′ untranslated region (5′UTR) in response to H. pylori infection in vitro. Methodology: Human cell lines (AGS, GES-1, HeLa, HEK293T) were used alongside bicistronic and monocistronic (Firefly/Renilla luciferases) reporter assays to assess short and long variants of the Survivin 5′UTR and HIV-1 internal ribosome entry site (IRES) sequences. Additional methods included in vitro transcription/translation, RT-qPCR, agarose gel electrophoresis, Western blotting, coupled/uncoupled translation assays, and siRNA silencing. Results: The short variant of the Survivin 5′ UTR supported cap-independent translation, like the HIV-1 IRES. Notably, H. pylori infection suppressed this translation in a GGT-dependent manner in gastric cells, and a similar reduction was observed following treatment with ATO, a known prooxidant. Conclusion: The Survivin 5′UTR exhibits cap-independent translation activity that is inhibited by H. pylori in a GGT-dependent manner, likely via oxidative stress. This mechanism helps to explain the downregulation of Survivin during gastric infection and indicates that oxidative stress can negatively affect both cellular and viral IRES-mediated translation. Full article

Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4⁺ fraction, activation, exhaustion, effector/terminal differentiation, and IFN-γ/TNF-α production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays. Full article