Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

[Preparation technology comparison and performance evaluation of different protein A affinity chromatographic materials].

Protein A affinity chromatographic materials are widely used in clinical medicine and biomedicine because of their specific interactions with immunoglobulin G (IgG). Both the characteristics of the matrix, such as its structure and morphology, and the surface modification method contribute to the affinity properties of the packing materials. The specific, orderly, and oriented immobilization of protein A can reduce its steric hindrance with the matrix and preserve its bioactive sites. In this study, four types of affinity chromatographic materials were obtained using agarose and polyglycidyl methacrylate (PGMA) spheres as substrates, and multifunctional epoxy and maleimide groups were used to fix protein A. The effects of the ethylenediamine concentration, reaction pH, buffer concentration, and other conditions on the coupling efficiency of protein A and adsorption performance of IgG were evaluated. Multifunctional epoxy materials were prepared by converting part of the epoxy groups of the agarose and PGMA matrices into amino groups using 0.2 and 1.6 mol/L ethylenediamine, respectively. Protein A was coupled to the multifunctional epoxy materials using 5 mmol/L borate buffer (pH 8) as the reaction solution. When protein A was immobilized on the substrates by maleimide groups, the agarose and PGMA substrates were activated with 25% (v/v) ethylenediamine for 16 h to convert all epoxy groups into amino groups. The maleimide materials were then converted into amino-modified materials by adding 3 mg/mL 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in dimethyl sulfoxide (DMSO) and then suspended in 5 mmol/L borate buffer (pH 8). The maleimide groups reacted specifically with the C-terminal of the sulfhydryl group of recombinant protein A to achieve highly selective fixation on both the agarose and PGMA substrates. The adsorption performance of the affinity materials for IgG was improved by optimizing the bonding conditions of protein A, such as the matrix type, matrix particle size, and protein A content, and the adsorption properties of each affinity material for IgG were determined. The column pressure of the protein A affinity materials prepared using agarose or PGMA as the matrix via the maleimide method was subsequently evaluated at different flow rates. The affinity materials prepared with PGMA as the matrix exhibited superior mechanical strength compared with the materials prepared with agarose. Moreover, an excellent linear relationship between the flow rate and column pressure of 80 mL/min was observed for this affinity material. Subsequently, the effect of the particle size of the PGMA matrix on the binding capacity of IgG was investigated. Under the same protein A content, the dynamic binding capacity of the affinity materials on the PGMA matrix was higher when the particle size was 44-88 µm than when other particle sizes were used. The properties of the affinity materials prepared using the multifunctional epoxy and maleimide-modified materials were compared by synthesizing affinity materials with different protein A coupling amounts of 1, 2, 4, 6, 8, and 10 mg/mL. The dynamic and static binding capacities of each material for bovine IgG were then determined. The prepared affinity material was packed into a chromatographic column to purify IgG from bovine colostrum. Although all materials showed specific adsorption selectivity for IgG, the affinity material prepared by immobilizing protein A on the PGMA matrix with maleimide showed significantly better performance and achieved a higher dynamic binding capacity at a lower protein grafting amount. When the protein grafting amount was 15.71 mg/mL, the dynamic binding capacity of bovine IgG was 32.23 mg/mL, and the dynamic binding capacity of human IgG reached 54.41 mg/mL. After 160 cycles of alkali treatment, the dynamic binding capacity of the material reached 94.6% of the initial value, indicating its good stability. The developed method is appropriate for the production of protein A affinity chromatographic materials and shows great potential in the fields of protein immobilization and immunoadsorption material synthesis.

Global transcriptome profiles provide insights into muscle cell development and differentiation on microstructured marine biopolymer scaffolds for cultured meat production.

Biomaterial scaffolds play a pivotal role in the advancement of cultured meat technology, facilitating essential processes like cell attachment, growth, specialization, and alignment. Currently, there exists limited knowledge concerning the creation of consumable scaffolds tailored for cultured meat applications. This investigation aimed to produce edible scaffolds featuring both smooth and patterned surfaces, utilizing biomaterials such as salmon gelatin, alginate, agarose and glycerol, pertinent to cultured meat and adhering to food safety protocols. The primary objective of this research was to uncover variations in transcriptomes profiles between flat and microstructured edible scaffolds fabricated from marine-derived biopolymers, leveraging high-throughput sequencing techniques. Expression analysis revealed noteworthy disparities in transcriptome profiles when comparing the flat and microstructured scaffold configurations against a control condition. Employing gene functional enrichment analysis for the microstructured versus flat scaffold conditions yielded substantial enrichment ratios, highlighting pertinent gene modules linked to the development of skeletal muscle. Notable functional aspects included filament sliding, muscle contraction, and the organization of sarcomeres. By shedding light on these intricate processes, this study offers insights into the fundamental mechanisms underpinning the generation of muscle-specific cultured meat.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Magnetic quaternary ammonium polymer bearing porous agarose for selective extraction of Aristolochic acids in the plasma.

Aristolochic acids (AAs) naturally occurring in the herbal genus Aristolochia are associated with a high risk of kidney failure, multiple tumors and cancers. However, approaches with high selectivity and rapidity for measuring AAs in biological samples are still inadequate. Inspired by the mechanism of AAs-induced nephrotoxicity, we designed a hybrid magnetic polymer-porous agarose (denoted as MNs@SiO2M@DNV-A), mimicking the effect of basic and aromatic residues of organic anion transporter 1 (OAT1) for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in the plasma. The monomers of vinylbenzyl trimethylammonium chloride (VBTAC), N-vinyl-2-pyrrolidinone (NVP) and divinylbenzene (DVB) were employed to construct the polymer layer, which provided a selective adsorption for AAs by multiple interactions. The porous agarose shell contributed to remove interfering proteins in the plasma samples. A magnetic solid-phase extraction (MSPE) based on the proposed composite enhanced the selectivity toward AA I and AA II in the plasma samples. In combination of HPLC analysis, the proposed method was proved to be applicable to fast and specific quantification of AAs in blood samples, which was characterized by a good linearity, high sensitivity, acceptable recovery, excellent repeatability and satisfactory reusability.

The α-linkage in funoran and agarose could be hydrolyzed by a GH96 family enzyme: Discovery of the α-funoranase.

Agarans represent a group of galactans extracted from red algae. Funoran and agarose are the two major types and commercially applied polysaccharides of agaran. Although the glycoside hydrolases targeting ß-glycosidic bonds of agaran have been widely investigated, those capable of degrading α-glycosidic bonds of agarose were limited, and the enzyme degrading α-linkages of funoran has not been reported till now. In this study, a GH96 family enzyme BiAF96A_Aq from a marine bacterium Aquimarina sp. AD1 was heterologously expressed in Escherichia coli. BiAF96A_Aq exhibited dual activities towards the characteristic structure of funoran and agarose, underscoring the multifunctionality of GH96 family members. Glycomics and NMR analysis revealed that BiAF96A_Aq hydrolyzed the α-1,3 glycosidic bonds between 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose-6-sulfate (G6S) of funoran, as well as LA and ß-d-galactopyranose (G) of agarose, through an endo-acting manner. The end products of BiAF96A_Aq were majorly composed of disaccharides and tetrasaccharides. The identification of the activity of BiAF96A_Aq on funoran indicated the first discovery of the funoran hydrolase for α-1,3 linkage. Considering the novel catalytic reaction, we proposed to name this activity as "α-funoranase" and recommended the assignment of a dedicated EC number for its classification.

Agarose-based 3D culture improved the developmental competence of oocyte-granulosa complex isolated from porcine preantral follicle.

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established. The present study indicated that the use of a modified medium with Ascorbic Acid (50 µM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3-0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac-H3-K9) and methylation of histone H3 at lysine 4 (Me-H3-K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.

Thermosensitive composite based on agarose and chitosan saturated with carbon dioxide. Preliminary study of requirements for production of new CSAG bioink.

This study introduces a method for producing printable, thermosensitive bioink formulated from agarose (AG) and carbon dioxide-saturated chitosan (CS) hydrogels. The research identified medium molecular weight chitosan as optimal for bioink production, with a preferred chitosan hydrogel content of 40-60 %. Rheological analysis reveals the bioink's pseudoplastic behavior and a sol-gel phase transition between 27.0 and 31.5 °C. The MMW chitosan-based bioink showed also the most stable extrusion characteristic. The choice of chitosan for the production of bioink was also based on the assessment of the antimicrobial activity of the polymer as a function of its molecular weight and the degree of deacetylation, noting significant cell reduction rates for E. coli and S. aureus of 1.72 and 0.54 for optimal bioink composition, respectively. Cytotoxicity assessments via MTT and LDH tests confirm the bioink's safety for L929, HaCaT, and 46BR.1 N cell lines. Additionally, XTT proliferation assay proved the stimulating effect of the bioink on the proliferation of 46BR.1 N fibroblasts, comparable to that observed with Fetal Bovine Serum (FBS). FTIR spectroscopy confirms the bioink as a physical polymer blend. In conclusion, the CS/AG bioink demonstrates the promising potential for advanced spatial cell cultures in tissue engineering applications including skin regeneration.

Dextrin-assisted gel electromembrane extraction of chiral drugs: Improving the extraction efficiency and investigation of enantioselectivity of extraction.

The present study investigates the use of dextrins (maltodextrin, ß-cyclodextrin, and hydroxypropyl-ß-cyclodextrin) to improve the efficiency of the agarose-based gel electromembrane extraction technique for extracting chiral basic drugs (citalopram, hydroxyzine, and cetirizine). Additionally, it examines the enantioselectivity of the extraction process for these drugs. To achieve these, dextrins were incorporated into either the sample solution, the membrane, or the acceptor solution, and then the extraction procedure was performed. Enantiomers were separated and analyzed using a capillary electrophoresis device equipped with a UV detector. The results obtained under the optimal extraction conditions (sample solution pH: 4.0, acceptor solution pH: 2.0, gel membrane pH: 3.0, agarose concentration: 3 % w/v, stirring rate: 1000 rpm, gel thickness: 4.4 mm, extraction voltage: 62.3 V, and extraction time: 32.1 min) indicated that incorporating dextrins into either the sample solution, membrane or the acceptor solution enhances extraction efficiency by 17.3-23.1 %. The most significant increase was observed when hydroxypropyl-ß-cyclodextrin was added to the acceptor solution. The findings indicated that the inclusion of hydroxypropyl-ß-cyclodextrin in the sample solution resulted in an enantioselective extraction, yielding an enantiomeric excess of 6.42-7.14 %. The proposed method showed a linear range of 5.0-2000 ng/mL for enantiomers of model drugs. The limit of detection and limit of quantification for all enantiomers were found to be < 4.5 ng/mL and <15.0 ng/mL, respectively. Intra- and inter-day RSDs (n = 4) were less than 10.8 %, and the relative errors were less than 3.2 % for all the enantiomers. Finally, the developed method was successfully applied to determine concentrations of enantiomers in a urine sample with relative recoveries of 96.8-99.2 %, indicating good reliability of the developed method.

Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Gelatine Blends Modified with Polysaccharides: A Potential Alternative to Non-Degradable Plastics.

Non-degradable plastics of petrochemical origin are a contemporary problem of society. Due to the large amount of plastic waste, there are problems with their disposal or storage, where the most common types of plastic waste are disposable tableware, bags, packaging, bottles, and containers, and not all of them can be recycled. Due to growing ecological awareness, interest in the topics of biodegradable materials suitable for disposable items has begun to reduce the consumption of non-degradable plastics. An example of such materials are biodegradable biopolymers and their derivatives, which can be used to create the so-called bioplastics and biopolymer blends. In this article, gelatine blends modified with polysaccharides (e.g., agarose or carrageenan) were created and tested in order to obtain a stable biopolymer coating. Various techniques were used to characterize the resulting bioplastics, including Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)/differential scanning calorimetry (DSC), contact angle measurements, and surface energy characterization. The influence of thermal and microbiological degradation on the properties of the blends was also investigated. From the analysis, it can be observed that the addition of agarose increased the hardness of the mixture by 27% compared to the control sample without the addition of polysaccharides. In addition, there was an increase in the surface energy (24%), softening point (15%), and glass transition temperature (14%) compared to the control sample. The addition of starch to the gelatine matrix increased the softening point by 15% and the glass transition temperature by 6%. After aging, both compounds showed an increase in hardness of 26% and a decrease in tensile strength of 60%. This offers an opportunity as application materials in the form of biopolymer coatings, dietary supplements, skin care products, short-term and single-contact decorative elements, food, medical, floriculture, and decorative industries.

Genipin crosslinking promotes biomechanical reinforcement and pro-regenerative macrophage polarization in bioartificial tubular substitutes.

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

Towards a biomimetic cellular structure and physical morphology with liposome-encapsulated agarose sol systems.

The cytoplasm, serving as the primary hub of cellular metabolism, stands as a pivotal cornerstone for the harmonious progression of life. The ideal artificial cell should not only have a biomembrane structure system similar to that of a cell and the function of carrying genetic information, but also should have an intracellular environment. In this pursuit, we employed a method involving the incorporation of glycerol into agarose, resulting in the formation of agarose-glycerol mixed sol (AGs). This dynamic sol exhibited fluidic properties at ambient temperature, closely mimicking the viscosity of authentic cytoplasm. Harnessing the electroformation technique, AGs was encapsulated within liposomes, enabling the efficient creation of artificial cells that closely resembled native cellular dimensions through meticulous parameter adjustments of the alternating current (AC) field. Subsequently, artificial cells harboring AGs were subjected to diverse electrolyte and non-electrolyte solutions, enabling a comprehensive exploration of their deformation phenomena, encompassing both inward and outward budding. This study represents a significant stride forward in addressing one of the most fundamental challenges in the construction of artificial cytoplasm. It is our fervent aspiration that this work shall offer invaluable insights and guidance for future endeavors in the realm of artificial cell construction.

Green synthesis of chitosan/glutamic acid/agarose/Ag nanocomposite hydrogel as a new platform for colorimetric detection of Cu ions and reduction of 4-nitrophenol.

In this study, the green synthesis of chitosan/glutamic acid/agarose/Ag (Chi/GA/Aga/Ag) nanocomposite hydrogel was obtained via in situ reduction of Ag ions during the crosslinking process of chitosan-agarose double network hydrogels. The rich hydroxyl, carboxyl and amino groups in both agarose, chitosan, and glutamic acid can effectively control the growth, dispersion and immobilization of nearly spherical Ag nanoparticles (70 nm) in the Chi/GA/Aga/Ag composite hydrogel. Glutamic acids can act as the structure-directing agents to induce the formation of chitosan/glutamic acid hydrogel. The mechanical strength of the Chi/GA/Aga/Ag composite hydrogel can be enhanced by the introduction of chitosan-agarose double network hydrogels, which guarantees that it can be directly used as a visual test strip of the Cu ions with a lower detection limit of 1 µM and an active catalyst for the reduction of 4-nitrophenol within 18 min. The quantitative and semi-quantitative measurement of Cu ions can be carried out by UV-visible absorption spectroscopy and visual measurement, which provided a convenient, portable, and "naked-eye" solid-state detection methodology.

Preparation and optimization of agarose or polyacrylamide/amino acid-based double network hydrogels for photocontrolled drug release.

The high water content and biocompatibility of amino-acid-based supramolecular hydrogels have generated growing interest in drug delivery research. Nevertheless, the existing dominant approach of constructing such hydrogels, the exploitation of a single amino acid type, typically comes with several drawbacks such as weak mechanical properties and long gelation times, hindering their applications. Here, we design a near-infrared (NIR) light-responsive double network (DN) structure, containing amino acids and different synthetic or natural polymers, i.e., polyacrylamide, poly(N-isopropylacrylamide), agarose, or low-gelling agarose. The hydrogels displayed high mechanical strength and high drug-loading capacity. Adjusting the ratio of Fmoc-Tyr-OH/Fmoc-Tyr(Bzl)-OH or Fmoc-Phe-OH/Fmoc-Tyr(Bzl)-OH, we could drastically shorten the gelation time of the DN hydrogels at room and body temperatures. Moreover, introducing photothermal agents (graphene oxide, carbon nanotubes, molybdenum disulfide nanosheets, or indocyanine green), we equipped the hydrogels with NIR responsivity. We demonstrated the light-triggered release of the drug baclofen, which is used in severe spasticity treatment. Rheology and stability tests confirmed the positive impact of the polymers on the mechanical strength of the hydrogels, while maintaining good stability under physiological conditions. Overall, our study contributed a novel hydrogel formulation with high mechanical resistance, rapid gel formation, and efficient NIR-controlled drug release, offering new opportunities for biomedical applications.

Characterization of a novel carbohydrate-binding module specifically binding to the major structural units of porphyran.

Porphyran is a promising bioactive polysaccharide majorly composed of 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) disaccharide repeating units. Carbohydrate-binding modules (CBMs) have been verified to be essential tools for investigating polysaccharides. However, no confirmed CBM binding to porphyran has been hitherto reported. In this study, an unknown domain with a predicted ß-sandwich fold from a potential GH86 porphyranase was discovered, and further recombinantly expressed. The CBM protein (named FvCBM99) presented a desired specificity for porphyran tetrasaccharide with an affinity constant of 1.9 × 10-4 M, while it could not bind to agarose tetrasaccharide. The sequence novelty and well-defined function of FvCBM99 and its homologs reveal a new CBM family, CBM99. Besides, the application potential of FvCBM99 in in situ visualization of porphyran was demonstrated. The discovery of FvCBM99 provides a favorable tool for future studies of porphyran.

Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells.

Cells with various structures and proteins naturally come together to cooperate in vivo. This study used cell spheroids cultured in agarose micro-wells as a 3D model to study the movement of cells or spheroids toward other spheroids. The formation dynamics of tumor spheroids and the interactions of two batches of cells in the agarose micro-wells were studied. The results showed that a concave bottom micro-well (diameter: 2 mm, depth: 2 mm) prepared from 3% agarose could be used to study the interaction of two batches of cells. The initial tumor cell numbers from 5 × 103 cells/well to 6 × 104 cells/well all could form 3D spheroids after 3 days of incubation. Adding the second batch of DU 145 cells to the existing DU 145 spheroid resulted in the formation of satellite cell spheroids around the existing parental tumor spheroid. Complete fusion of two generation cell spheroids was observed when the parental spheroids were formed from 1 × 104 and 2 × 104 cells, and the second batch of cells was 5 × 103 per well. A higher amount of the second batch of cells (1 × 104 cell/well) led to the formation of independent satellite spheroids after 48 h of co-culture, suggesting the behavior of the second batch of cells towards existing parental spheroids depended on various factors, such as the volume of the parental spheroids and the number of the second batch cells. The interactions between the tumor spheroids and Human Umbilical Vein Endothelial Cells (HUVECs) were modeled on concave agarose micro-wells. The HUVECs (3 × 103 cell/well) were observed to gather around the parental tumor spheroids formed from 1 × 104, 2 × 104, and 3 × 104 cells per well rather than aggregate on their own to form HUVEC spheroids. This study highlights the importance of analyzing the biological properties of cells before designing experimental procedures for the sequential fusion of cell spheroids. The study further emphasizes the significant roles that cell density and the volume of the spheroids play in determining the location and movement of cells.

Cyclic deformation behavior of agarose hydrogels prepared at different gelation concentrations.

Effects of the gelation concentration (c0) on cyclic deformation behavior of agarose hydrogels have been examined. Agarose hydrogels prepared at c0 = 10 g L-1 (A10) show smaller Young's modulus than those prepared at c0 = 30 g L-1 (A30) even when the mechanical properties have been measured at the same agarose concentration 50 g L-1. Moreover, A10 has stronger tendency to exhibit the residual strain in the cyclic deformation curves compared to A30. These results can be explained based on the gelation mechanism specific to agarose, where helices of agarose aggregate via hydrogen bonding to be the crosslinks. It has been proposed from the optical rotation that the helix content just after gelation increases with c0, while the helix content for A10 was higher than that for A30 at 50 g L-1. Since the lower helix content at c0 for A10 results in less stable aggregates of agarose helices, it can be said that the cyclic deformation behavior as well as Young's modulus of agarose hydrogels primarily reflects the network structure determined by c0.

Encapsulating therapeutic cells in RGD-modified agarose microcapsules.

Current cell-based strategies for repairing damaged tissue often show limited efficacy due to low cell retention at the site of injury. Encapsulation of cells within hydrogel microcapsules demonstrably increases cell retention but benefits can be limited due to premature cell escape from the hydrogel microcapsules and subsequent clearance from the targeted tissue. We propose a method of encapsulating cells in agarose microcapsules that have been modified to increase cell retention by providing cell attachment domains within the agarose hydrogel allowing cells to adhere to the microcapsules. We covalently modified agarose with the addition of the cell adhesion peptide, RGD (arginine, glycine, aspartic acid). We then used a microfluidic platform to encapsulate single cells within 50 µm agarose microcapsules. We tracked encapsulated cells for cell viability, egress from microcapsules and attachment to microcapsules at 2 h, 24 h, and 48 h after encapsulation. Many encapsulated cells eventually egress their microcapsule. Those that were encapsulated using RGD-modified agarose adhered to the outer surface of the microcapsule following egress. NIH 3T3 cells showed nearly 45% of egressed cells attached to the outside of RGD modified agarose microcapsules, while minimal cellular adhesion was observed when using unmodified agarose. Similarly, human umbilical vein endothelial cells had up to 33% of egressed cells attached and explant-derived cardiac cells showed up to 20% attachment with the presence of RGD binding domains within the agarose microcapsules.

Optimization of the Composition of Mesoporous Polymer-Ceramic Nanocomposite Granules for Bone Regeneration.

Difficult-to-treat bone damage resulting from metabolic bone diseases, mechanical injuries, or tumor resection requires support in the form of biomaterials. The aim of this research was to optimize the concentration of individual components of polymer-ceramic nanocomposite granules (nanofilled polymer composites) for application in orthopedics and maxillofacial surgery to fill small bone defects and stimulate the regeneration process. Two types of granules were made using nanohydroxyapatite (nanoHA) and chitosan-based matrix (agarose/chitosan or curdlan/chitosan), which served as binder for ceramic nanopowder. Different concentrations of the components (nanoHA and curdlan), foaming agent (sodium bicarbonate-NaHCO3), and chitosan solvent (acetic acid-CH3COOH) were tested during the production process. Agarose and chitosan concentrations were fixed to be 5% w/v and 2% w/v, respectively, based on our previous research. Subsequently, the produced granules were subjected to cytotoxicity testing (indirect and direct contact methods), microhardness testing (Young's modulus evaluation), and microstructure analysis (porosity, specific surface area, and surface roughness) in order to identify the biomaterial with the most favorable properties. The results demonstrated only slight differences among the resultant granules with respect to their microstructural, mechanical, and biological properties. All variants of the biomaterials were non-toxic to a mouse preosteoblast cell line (MC3T3-E1), supported cell growth on their surface, had high porosity (46-51%), and showed relatively high specific surface area (25-33 m2/g) and Young's modulus values (2-10 GPa). Apart from biomaterials containing 8% w/v curdlan, all samples were predominantly characterized by mesoporosity. Nevertheless, materials with the greatest biomedical potential were obtained using 5% w/v agarose, 2% w/v chitosan, and 50% or 70% w/v nanoHA when the chitosan solvent/foaming agent ratio was equal to 2:2. In the case of the granules containing curdlan/chitosan matrix, the most optimal composition was as follows: 2% w/v chitosan, 4% w/v curdlan, and 30% w/v nanoHA. The obtained test results indicate that both manufactured types of granules are promising implantable biomaterials for filling small bone defects that can be used in maxillofacial surgery.