An enhanced and rapid method for von Willebrand factor multimer analysis for mechanical circulatory device testing.

BACKGROUND: Von Willebrand factor (VWF) is a critical glycoprotein in hemostasis and is an important factor in diagnosing bleeding disorders. Albeit the analysis of VWF is often compromised by inconsistent methodologies and challenges quantifying multimeric size. Current VWF multimer analysis methods are costly, time-consuming, and often inconsistent; thus, demanding skilled professionals. This study aimed to streamline and optimize the VWF multimer analysis technique, making it more efficient and reproducible, particularly for identifying or predicting mechanical circulatory support (MCS) induced bleeding disorders. METHODS: Blood samples from healthy volunteers were exposed to high shear forces via a Medtronic HeartWare ventricular assist device. VWF multimers were analyzed using vertical-gel agarose electrophoresis and Western blotting. Differences in VWF distribution were determined using densitometry, and two methods of densitometric analysis were compared: proprietary software against open-source software. RESULTS: Using the developed method: (i) protocol duration was accelerated from three days (in classical methods) to ~ eight hours; (ii) the resolution of the high molecular weight (HMW) VWF multimers were substantially improved; and (iii) densitometric analysis tools were validated. Additionally, the densitometry analysis using two software types showed a strong correlation between results, with the proprietary software reporting slightly higher HMW VWF percentages. CONCLUSION: This methodology is recommended for affordable, accurate, and reproducible VWF multimer evaluations during MCS use and testing. Further research comparing this method with semi-automated methods would provide additional insight and improve inter-laboratory comparisons.

Fabrication of GelMA - Agarose Based 3D Bioprinted Photocurable Hydrogel with In Vitro Cytocompatibility and Cells Mirroring Natural Keratocytes for Corneal Stromal Regeneration.

The complex anatomy of the cornea and the subsequent keratocyte-fibroblast transition have always made corneal stromal regeneration difficult. Recently, 3D printing has received considerable attention in terms of fabrication of scaffolds with precise dimension and pattern. In the current work, 3D printable polymer hydrogels made of GelMA/agarose are formulated and its rheological properties are evaluated. Despite the variation in agarose content, both the hydrogels exhibited G'>G'' modulus. A prototype for 3D stromal model is created using Solid Works software, mimicking the anatomy of an adult cornea. The fabrication of 3D-printed hydrogels is performed using pneumatic extrusion. The FTIR analysis speculated that the hydrogel is well crosslinked and established strong hydrogen bonding with each other, thus contributing to improved thermal and structural stability. The MTT analysis revealed a higher rate of cell proliferation on the hydrogels. The optical analysis carried out on the 14th day of incubation revealed that the hydrogels exhibit transparency matching with natural corneal stromal tissue. Specific protein marker expression confirmed the keratocyte phenotype and showed that the cells do not undergo terminal differentiation into stromal fibroblasts. The findings of this work point to the potential of GelMA/A hydrogels as a novel biomaterial for corneal stromal tissue engineering.

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Three-Dimensionally Printed Agarose Micromold Supports Scaffold-Free Mouse Ex Vivo Follicle Growth, Ovulation, and Luteinization.

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery.

Enhanced mechanical properties and biocompatibility of hydroxyapatite scaffolds by magnesium and titanium oxides for bone tissue applications.

Significant attention has been devoted to bioactive implants for bone tissue applications, particularly composite scaffolds based on hydroxyapatite (HaP). This study explores the effects of Magnesium and Titanium oxides on the characteristics of HaP-based composite (HMT) scaffolds. The ceramic nanopowders were synthesized using in situ sol-gel, and then the scaffolds were fabricated by gel-casting technique, followed by heat treatment at 1200 °C. The thermal, microstructural, and structural properties of the samples were investigated by different characterization techniques. It was observed that the formation of the MgTiO3 phase in the composite scaffold was likely the key factor contributing to the improved mechanical properties. Finally, to evaluate bioactivity and biodegradability, scaffolds were immersed in simulated body fluid (SBF) buffer and analyzed by Field Emission Scanning Electron Microscopy (FESEM), and the viability of human fibroblast cells was assessed using the MTT assay. The composite scaffolds containing the MgTiO3 phase showed greater HaP layer formation on the scaffold surface, indicating enhanced biocompatibility.

Fabrication of micron-sized boronate-decorated polyethyleneimine-grafted magnetic agarose beads for specific enrichment of ribonucleic acid.

Exploiting high-performance magnetic beads for specific enrichment of ribonucleic acid (RNA) has important significance in the biomedical research field. Herein, a simple strategy was proposed for fabricating boronate-decorated polyethyleneimine-grafted magnetic agarose beads (BPMAB), which can selectively isolate cis-diol-containing substances through boronate affinity. The size of the basic magnetic agarose beads was controlled through the emulsification of the water-in-oil emulsion with a high-speed shear machine, which enhanced the specific surface area of BPMAB. Subsequently, to modify more boronic acid ligands, branched PEI with excellent hydrophilicity and numerous reaction sites was grafted. 2,4-Difluoro-3-formylphenyl boronic acid (2,4-DFPBA) was covalently immobilized for selectively capturing cis-diol-containing substances under physiological condition (pH 7.4). The BPMAB with a diameter range from 1.86 µm to 11.60 µm possessed clearly spherical structure, and excellent magnetic responsiveness and suspension ability in aqueous solution. ß-Nicotinamide adenine dinucleotide (ß-NAD), a short-chain cis-diol carrying agent, was selected as a target molecule for evaluating the adsorption property of BPMAB and the maximum adsorption capacity of BPMAB for ß-NAD could reach 205.11 mg g-1. In addition, the BPMAB as adsorbent was used to selectively enrich RNA from mammalian cells. The maximum adsorption capacity of BPMAB for RNA was 140.50 mg g-1. Under optimized conditions, the BPMAB-based MSPE successfully enriched the high-quality total RNA with 28S to 18S ribosomal RNA ratios ranging from 2.06 to 2.16. According to the PCR analysis of GADPH gene, the extracted total RNA was successfully reverse transcribed into cDNA. Therefore, we believe that the BPMAB-based MSPE could be applicable for the specific enrichment of RNA from complex biological systems.

PEG-modified Fe2O3 coated agarose hydrogel: A synthesized nanocomposite for regulated 5-fluorouracil delivery.

An innovative pH-responsive nanocomposite, comprising agarose (AGA) modified with polyethylene glycol (PEG) hydrogel and coated with ferric oxide (Fe2O3), has been formulated to facilitate the precise administration of 5-fluorouracil (5-Fu) to breast cancer cells. By utilizing a double emulsion technique, the size of the nanocomposites was significantly reduced through the application of almond oil; the inclusion of span 80 further improved their uniformity. The physiochemical properties of the nanocomposite were thoroughly examined by Fourier Transformed Infrared (FT-IR), X-ray diffraction (XRD), Field Emission-Scanning Electron Microscope (FE-SEM), Vibrating Sample Magnetometer (VSM), dynamic light scattering (DLS), and zeta potential tests. The verification of the uniform particle distribution was achieved by employing FE-SEM and VSM analyses. The average diameter of the particles was 223 nm, and their zeta potential was -47.6 mV. In addition, the nanocomposite exhibited a regulated release of 5-Fu at pH 5.4 and pH 7.4, as indicated by an in vitro drug release profile. PEG-AGA- Fe2O3@5-Fu exhibited biocompatibility, as indicated by the lack of deleterious effects observed in tumor cells. This revolutionary nanocomposite demonstrates exceptional promise for breast cancer treatment, underscoring its significance as a major advancement in the pursuit of novel nanotechnologies for cancer therapy.

Preparation and characterization of agarose-sodium alginate hydrogel beads for the co-encapsulation of lycopene and resveratrol nanoemulsion.

In the study, lycopene and resveratrol nanoemulsion hydrogel beads were prepared by using agarose­sodium alginate as a carrier and the semi-interpenetrating polymer network technique, characteristics and morphologies were evaluated by scanning electron microscopy, fluorescence microscopy, rheological measurement. The synergistic antioxidant effect of lycopene and resveratrol was confirmed, the best synergistic antioxidant performance is achieved when the ratio of 1:1. To increase the solubility and improve the stability, the lycopene was prepared as solid dispersion added to the nanoemulsion. The encapsulation rate of lycopene and resveratrol reached 93.60 ± 2.94 % and 89.30 ± 1.75 %, respectively, and the cumulative release showed that the addition of agarose slowed down the release rate of the compound, which improves the applicability of lycopene and resveratrol and development of carriers for the delivery of different bioactive ingredients.

Non-specific markers of inflammation in bare-nosed wombats (Vombatus ursinus) with sarcoptic mange.

Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei, negatively impacts the health, welfare, and local abundance of bare-nosed wombats (Vombatus ursinus) in Australia. Improved understanding of the host immune response to disease and its contribution to pathophysiology could be used to inform management actions for this species in and ex situ. To evaluate the immune response of bare-nosed wombats to sarcoptic mange, we validated three assays (haptoglobin, agarose gel electrophoresis, and micro-erythrocyte sedimentation rate) measuring non-specific markers of inflammation using serum samples from free-living wombats from Tasmania (n = 33). We then analysed correlations between the assay results for each non-specific marker of inflammation and wombat's sarcoptic mange scores, and performed histopathological examinations to investigate association of the acute phase response with systemic amyloidosis. We present evidence that haptoglobin and erythrocyte sedimentation rate increased, and albumin decreased, in association with sarcoptic mange scores. This research demonstrates links between the acute phase response and sarcoptic mange severity in bare-nosed wombats, highlighting the utility of non-specific markers of inflammation for aiding assessment of the systemic effects of mange. Showing the value of agarose gel electrophoresis, we also identified specific acute phase proteins warranting future evaluation and found evidence of an immunoglobulin response in mange-affected wombats, revealed by increasing γ-globulins in association with apparent disease severity. Meanwhile, owing to its relatively low resource requirements and rapidity, the erythrocyte sedimentation rate assay may be useful as a point-of-care test to support therapeutic decisions in the field. Our methods and findings are likely to be applicable to a range of other clinical and population health scenarios in captive and free-living wombats, and species impacted by sarcoptic mange globally.

Production of Agarose-Hydroxyapatite Composites via Supercritical Gel Drying, for Bone Tissue Engineering.

Bone tissue engineering (BTE) is the most promising strategy to repair bones injuries and defects. It relies on the utilization of a temporary support to host the cells and promote nutrient exchange (i.e., the scaffold). Supercritical CO2 assisted drying can preserve scaffold nanostructure, crucial for cell attachment and proliferation. In this work, agarose aerogels, loaded with hydroxyapatite were produced in view of BTE applications. Different combinations of agarose concentration and hydroxyapatite loadings were tested. FESEM and EDX analyses showed that scaffold structure suffered from partial closure when increasing filler concentration; hydroxyapatite distribution was homogenous, and Young's modulus improved. Looking at BTE applications, the optimal combination of agarose and hydroxyapatite resulted to be 1% w/w and 10% w/v, respectively. Mechanical properties showed that the produced composites could be eligible as starting scaffold for BTE, with a Young's Modulus larger than 100 kPa for every blend.

Layered complexity, reorganisational ability and self-healing mechanisms of heteropolysaccharide solutions.

Heteropolysaccharides are among the most widely distributed compounds in nature, acting as both tissue building blocks and as a source of nutrients. Their physicochemical and biological properties have been studied thoroughly; however, the microstructural properties of heteropolysaccharides are still poorly understood. This study aims to investigate the micro-structural peculiarities of agarose, gum arabic, hyaluronic and alginic acids by means of confocal laser scanning microscopy (CLSM) and cryogenic scanning electron microscopy (cryo-SEM). Herein, attention is paid to layered complexity of the microstructure differentiating surface, under surface, inner, and substrate interface layers. The scale and pattern of the polysaccharide's microstructure depend on the concentration, changing from lamellae to cell-like porous structures. This work provides the insight into micro- and nanoscale mechanisms of self-healing and substrate-induced reorganisation. Thus, investigation of the self-healing mechanism revealed that this diffusion-based process starts from the fibres, turning into lamellae, following by cell-like structures with smaller dimensions. Investigation of the substrate-induced reorganisation ability showed that nano-to-micro (scale) porous substrate causes reorganisation in the interface layer of the studied heteropolysaccharides. This work contributes to understanding the structural peculiarities of heteropolysaccharides by looking at them through a supramolecular, micro-level prism.

Agarose as a Tissue Mimic for the Porcine Heart, Kidney, and Liver: Measurements and a Springpot Model.

Agarose gels are often used as a tissue mimic. The goal of this work was to determine the appropriate agarose concentrations that result in mechanical properties that match three different porcine organs. Strain tests were carried out with an amplitude varying from 0.01% to 10% at a frequency of 1 Hz on a range of agarose concentrations and porcine organs. Frequency sweep tests were performed from 0.1 Hz to a maximum of 9.5 Hz at a shear strain amplitude of 0.1% for agarose and porcine organs. In agarose samples, the effect of pre-compression of the samples up to 10% axial strain was considered during frequency sweep tests. The experimental measurements from agarose samples were fit to a fractional order viscoelastic (springpot) model. The model was then used to predict stress relaxation in response to a step strain of 0.1%. The prediction was compared to experimental relaxation data, and the results agreed within 12%. The agarose concentrations (by mass) that gave the best fit were 0.25% for the liver, 0.3% for the kidney, and 0.4% for the heart. At a frequency of 0.1 Hz and a shear strain of 0.1%, the agarose concentrations that best matched the shear storage modulus of the porcine organs were 0.4% agarose for the heart, 0.3% agarose for the kidney, and 0.25% agarose for the liver.

A New Capillary-Channel Agarose Sponge Assembled with Deep Eutectic Solvent Based Magnetic Fluid for Rapid Hemostasis and Prevention of Bacterial Infection.

A new composite sponge assisted by magnetic field-mediated guidance was developed for effective hemostasis. It was based on polydopamine capillary-channel agarose (PDA-CAGA) sponge as matrix; meanwhile, the combination of deep eutectic solvent (DES, choline chloride:glycerol = 1:1, M/M)-dispersed Fe3O4 nanoparticles after fabrication by tannic acid (DES-Fe3O4@TA) was applied as hemostatic magnetic fluid. This sponge had oriented and aligned capillary channels realized by a 3D printed pattern, which endowed them with obvious shape memory and liquid absorption performance. Computational simulation was performed to describe the fluid status in channels; DES-Fe3O4@TA exhibited good magnetic properties, fluidity, and stability. In addition, the sponge driven to react rapidly with the bleeding site under the effect of a magnetic field presented a shorter hemostasis time (reduced by 85.02% in the tail and 81.07% in the liver of rats) and less blood loss (reduced by 97.08% in the tail and 91.50% in the liver) than those of medical gelatin sponge (GS). Meanwhile, the multifunctional material also exhibited better biocompatibility, procoagulant performance, and significant inhibition on S. aureus and E. coli than GS. As a whole, this work proposed a new strategy for rapid hemostasis by designing a magnetic field assisted composite bacteriostatic material, which also expanded the applications of green solvents in the clinical management field.

UGT1A1*28 detection using high-resolution agarose gel electrophoresis.

A new UGT1A1*28 detection method combining PCR and high-resolution agarose gel electrophoresis was developed. The viability of this method was demonstrated on 15 healthy adult volunteers. Subjects included 13 wild type homozygotes (86.7 %), 2 heterozygotes (13.3 %), and no mutant type homozygotes (0 %). The new UGT1A1*28 detection method results were fully consistent with DNA sequencing. PCR and agarose gel electrophoresis are common techniques with high-resolution agarose gels available commercially. These results support the clinical viability of this method potentially reducing UGT1A1*28 diagnosis complexity and cost.

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.

Age, sex and breed effect on laboratory parameters in natural Babesia canis infection.

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), ß- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

Preparation of a sustainable magnetic sorbent for the extraction and preconcentration of progestogens in natural water samples.

A film composed of agarose and graphene (G) and magnetic nanoparticles (G-MNPs) is proposed as a sorbent for the extraction and determination of medroxyprogesterone (MED), levonorgestrel (LEV), norethisterone (NOR) and progesterone (PRO) in natural water samples. Both the preparation of the film and the extraction procedure were optimized. The optimal extraction parameters were as follows: isopropyl alcohol as activation solvent, sample pH value of 3.0, extraction time of 30 min, 1.00 mL of acetonitrile as eluent, elution time of 5 min and sample volume of 100.00 mL. HPLC with photodiode array detector was used for the separation and determination. The method presented a linear range between 2.50 and 75.0 µg L-1 for all analytes, and the LODs were between 1.40 and 1.80 µg L-1. The method was applied to natural water samples, obtaining satisfactory recovery values (75-111 %). In conclusion, for the immobilization of the G-MNPs, agarose was used, which is a non-toxic, renewable and biodegradable material. The G-MNPs-agarose film was reused up to 70 times, without losing its extraction capacity significantly and presenting excellent sorbent properties, which allow the extraction and preconcentration of the progestogens under study.

Woven Agarose-Cellulose Composite Aerogel Fibers with Outstanding Radial Elasticity for Personal Thermal Management.

Aerogel fibers are good thermal insulators, suitable for weaving, and show potential as the next generation of intelligent textiles that can effectively reduce heat consumption for personal thermal management. However, the production of continuous aerogel fibers from biomass with sufficient strength and radial elasticity remains a significant challenge. Herein, continuous gel fibers were produced via wet spinning using agarose (AG) as the matrix, 2,2,2,6,6-tetramethylpiperidine-1-oxyl radical-oxidized cellulose nanofibers (TOCNs) as the reinforcing agent, and no other chemical additives by utilizing the gelling properties of AG. Supercritical drying and chemical vapor deposition (CVD) were then used to produce hydrophobic AG-TOCN aerogel fibers (HATAFs). During CVD, the HATAF gel skeleton was covered with an isostructural silica coating. Consequently, the HATAFs can recover from radial compression under 60% strain. Moreover, the HATAFs have low densities (≤0.14 g cm-3), high porosities (≥91.8%), high specific surface areas (≥188 m2 g-1), moderate tensile strengths (≤1.75 MPa), excellent hydrophobicity (water contact angles of >130°), and good thermal insulating properties at different temperatures. Thus, HATAFs are expected to become a new generation of materials for efficient personal thermal management.

Methylation-GC-MS/FID-Based Glycosidic Linkage Analysis of Unfractionated Polysaccharides in Red Seaweeds.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

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.