Microwave-assisted synthesis of highly sulfated mannuronate glycans as potential inhibitors against SARS-CoV-2.

Algae-based marine carbohydrate drugs are typically decorated with negative ion groups such as carboxylate and sulfate groups. However, the precise synthesis of highly sulfated alginates is challenging, thus impeding their structure-activity relationship studies. Herein we achieve a microwave-assisted synthesis of a range of highly sulfated mannuronate glycans with up to 17 sulfation sites by overcoming the incomplete sulfation due to the electrostatic repulsion of crowded polyanionic groups. Although the partially sulfated tetrasaccharide had the highest affinity for the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, the fully sulfated octasaccharide showed the most potent interference with the binding of the RBD to angiotensin-converting enzyme 2 (ACE2) and Vero E6 cells, indicating that the sulfated oligosaccharides might inhibit the RBD binding to ACE2 in a length-dependent manner.

In vitro effects of wound-dressings on key wound healing properties of dermal fibroblasts.

Healing of complex wounds requires dressings that must, at least, not hinder and should ideally promote the activity of key healing cells, in particular fibroblasts. This in vitro study assessed the effects of three wound-dressings (a pure Ca2+ alginate: Algostéril®, a Ca2+ alginate + carboxymethylcellulose: Biatain alginate® and a polyacrylate impregnated with lipido-colloid matrix: UrgoClean®) on dermal fibroblast activity. The results showed the pure calcium alginate to be non-cytotoxic, whereas the other wound-dressings showed moderate to strong cytotoxicity. The two alginates stimulated fibroblast migration and proliferation, whereas the polyacrylate altered migration and had no effect on proliferation. The pure Ca2+ alginate significantly increased the TGF-ß-induced fibroblast activation, which is essential to healing. This activation was confirmed by a significant increase in Vascular endothelial growth factor (VEGF) secretion and a higher collagen production. The other dressings reduced these fibroblast activities. The pure Ca2+ alginate was also able to counteract the inhibitory effect of NK cell supernatants on fibroblast migration. These in vitro results demonstrate that tested wound-dressings are not equivalent for fibroblast activation. Only Algostéril was found to promote all the fibroblast activities tested, which could contribute to its healing efficacy demonstrated in the clinic.

Interpenetrating gelatin/alginate mixed hydrogel: The simplest method to prepare an autoclavable scaffold.

The choice of sterilization method for hydrogels used for cell culture influences the ease of preparing the gel. We prepared interpenetrating gelatin/calcium alginate hydrogels containing 1% (w/v) alginate and 1-16% (w/v) gelatin by molding with the mixture of gelatin/sodium alginate solution, followed by the addition of calcium ions by incubation in calcium chloride solution. It is the simplest method to prepare autoclavable gelatin/sodium hydrogel. We measured various properties of the hydrogels including volume, Young's modulus in the compression test, storage modulus, and loss modulus in the dynamic viscoelasticity measurement. The gelatin/alginate hydrogel can be easily fabricated into any shape by this method. After autoclave treatment, the hydrogel was shrunk to smaller than the original shape in similar figures. The shape of the gelatin/alginate hydrogel can be designed into any shape with the reduction ratio of the volume. Human osteosarcoma (HOS) cells adhered to the gelatin/alginate hydrogel and then proliferated. Gelatin/calcium alginate hydrogels with a high concentration are considered to be autoclavable culture substrates because of their low deformation and gelatin elution rate after autoclaving and the high amount of cells attached to the hydrogels.

Physicochemical Properties and Biological Activities of Quinoa Polysaccharides.

Quinoa, known as the "golden grain" for its high nutritional value, has polysaccharides as one of its sources of important nutrients. However, the biological functions of quinoa polysaccharides remain understudied. In this study, two crude polysaccharide extracts of quinoa (Q-40 and Q-60) were obtained through sequential precipitation with 40% and 60% ethanol, with purities of 58.29% (HPLC) and 62.15% (HPLC) and a protein content of 8.27% and 9.60%, respectively. Monosaccharide analysis revealed that Q-40 contained glucose (Glc), galacturonic acid (GalA), and arabinose (Ara) in a molar ratio of 0.967:0.027:0.006. Q-60 was composed of xylose (xyl), arabinose (Ara), galactose, and galacturonic acid (GalA) with a molar ratio of 0.889:0.036:0.034:0.020. The average molecular weight of Q-40 ranged from 47,484 to 626,488 Da, while Q-60 showed a range of 10,025 to 47,990 Da. Rheological experiments showed that Q-40 exhibited higher viscosity, while Q-60 demonstrated more elastic properties. Remarkably, Q-60 showed potent antioxidant abilities, with scavenging rates of 98.49% for DPPH and 57.5% for ABTS. Antibacterial experiments using the microdilution method revealed that Q-40 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), while Q-60 specifically inhibited MRSA. At lower concentrations, both polysaccharides inhibited MDA (MD Anderson Cancer Center) cell proliferation, but at higher concentrations, they promoted proliferation. Similar proliferation-promoting effects were observed in HepG2 cells. The research provides important information in the application of quinoa in the food and functional food industries.

All-aqueous droplets-templated tailorable core-shell alginate microspheres for constructing vascularized intestinal mucosain vitromodels.

Recently,in vitromodels of intestinal mucosa have become important tools for drug screening and studying the physiology and pathology of the intestine. These models enable the examination of cellular behavior in diseased states or in reaction to alterations in the microenvironment, potentially serving as alternatives to animal models. One of the major challenges in constructing physiologically relevantin vitromodels of intestinal mucosa is the creation of three-dimensional microstructures that accurately mimic the integration of intestinal epithelium and vascularized stroma. Here, core-shell alginate (Alg) microspheres were generated to create the compartmentalized extracellular matrix microenvironment needed to simulate the epithelial and vascularized stromal compartments of the intestinal mucosa. We demonstrated that NIH-3T3 and human umbilical vein endothelial cells embedded in the core of the microspheres can proliferate and develop a vascular network, while human colorectal adenocarcinoma cells (Caco-2) can form an epithelial monolayer in the shell. Compared to Caco-2 monolayer encapsulated within the shell, the presence of the vascularized stroma enhances their proliferation and functionality. As such, our core-shell Alg microspheres provide a valuable method for generatingin vitromodels of vascularized intestinal mucosa with epithelial and vascularized stroma arranged in a spatially relevant manner and demonstrating near-physiological functionality.

A novel, microfluidic high-throughput single-cell encapsulation of human bone marrow mesenchymal stromal cells.

The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads' size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells' ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.

Evaluation of citrus pectin extraction methods: Synergistic enhancement of pectin's antioxidant capacity and gel properties through combined use of organic acids, ultrasonication, and microwaves.

The biological potency of pectin is intricately intertwined with its intricate molecular architecture. The fine structure of pectin is influenced by the extraction method, while the specific impact of these methods on the fine structure and the affected attributes thereof remains enigmatic. This study delves into the profound analysis of eight distinct extraction methods influence on the structure and biological activity of citrus peel pectin. The findings demonstrate that citric acid ultrasound-assisted microwave extraction yields pectin (PectinCA-US/MV) with higher viscosity and a dense, rigid chain. Pectin extracted with acetic acid ultrasound (PectinAA-US) and citric acid ultrasound (PectinCA-US) exhibits elevated galacturonic acid (GalA) levels and reduced D-galactose (Gal) content, enhancing antioxidant activity. Eight pectin-chitosan (CS) hydrogels, especially PectinCA-US/MV-CS, demonstrate commendable thermal stability, rheological properties, self-healing capability, and swelling behavior. This study characterizes citrus peel pectin properties from different extraction methods, laying a foundation for its application in food, pharmaceuticals, and industry.

Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Development and characterization of letrozole-encapsulated polymeric beads for sustained release.

The study aimed to prepare and characterize biodegradable sustained-release beads of letrozole (LTZ) for treating cancerous disease. The ionotropic gelation method was used for the preparation and calcium chloride (CaCl2) was used as a gelating agent, while chitosan (CTS) and sodium alginate (NaAlg) as biodegradable polymeric matrices in the blend hydrogel beads. The beads were characterized for their size, surface morphology, drug entrapment efficiency, drug-polymer interaction and crystallinity using different analytic techniques, including optical microscopy, Scanning Electron Microscopy (SEM), UV-spectroscopy, Fourier-transform Infrared Spectroscopy (FTIR), Thermo gravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and X-ray Diffraction Analysis (XRD) respectively. In vitro swelling studies were also applied to observe the response of these polymeric networks against different pH (at 1.2, 6.8 and 7.4 pH). The results from TGA and DSC exhibited that the components in the formulation possess better thermal stability. The XRD of polymeric networks displays a minor crystalline and significant amorphous nature. The SEM micrographs revealed that polymeric networks have uneven surfaces and grooves. Better swelling and in vitro outcomes were achieved at a high pH (6.8,7.4), which endorsed the pH-responsive characteristics of the prepared beads. Hence, beads based on chitosan and sodium alginate were successfully synthesized and can be used for the controlled release of letrozole.

Alginate-chitosan-microencapsulated tyrosols/oleuropein-rich olive mill waste extract for lipopolysaccharide-induced skin fibroblast inflammation treatment.

The Ca2+ ion-driven emulsification-ionotropic gelation method produced chitosan-alginate microspheres (CAMSs) with a narrow particle size distribution (PSD). Particle size distribution and zeta potential studies, as well as spectral electron microscopy, were used to assess the microspheres' physicochemical properties and morphology. The tyrosols (hydroxytyrosol (HT), tyrosol (TY), and oleuropein (OE) were loaded into these microspheres using a polyphenol extract (PPE) from Koroneki olive mill waste (KOMW). The microencapsulation efficiency and loading capacity of microspheres for PPE were 98.8% and 3.9%, respectively. Three simulated fluids, including gastric (pH = 1.2), intestinal (pH = 6.8), and colonic (pH = 7.4), were used to examine how the pH of the releasing medium affected the ability of CAMSs to release bioactive phenols. At a severely acidic pH (1.2, SGF), PPE release is nearly halted, while at pH 6.8 (SCF), release is at its maximum. Additionally, the PPE-CAMPs have ameliorated the endogenous antioxidant content SOD, GST, GPx with significant values from 0.05 to 0.01 in the treated LPS/human skin fibroblast cells. The anti-inflammatory response was appeared through their attenuations activity for the released cytokines TNF-α, IL6, IL1ß, and IL 12 with levels significantly from 0.01 to 0.001. Microencapsulation of PPE by CAMPs significantly improved its antioxidant and anti-inflammatory capabilities.

Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid.

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.

Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Preparation, characterization and release kinetics of a multilayer encapsulated Perilla frutescens L. essential oil hydrogel bead.

Encapsulation has been widely used as the protection of essential oils, which gives the possibility of their implementation as food preservatives. In this study, Perilla frutescens L. essential oil (PLEO) microcapsule powders were prepared firstly by spray drying method using octenyl succinic anhydride starch (OSAs) as wall material, and then they were further encapsulated by sodium alginate and chitosan via polyelectrolyte complex coacervates method. The best results were obtained by using 4 % of OSAs-PLEO microcapsule powders, 2 % of sodium alginate and 1.5 % of chitosan producing PLEO hydrogel beads with encapsulation efficiency of 61.29 % and loading degree of 41.11 %. Morphology observation showed PLEO hydrogel beads was a millimeter scale spherical particle. FTIR assay confirmed the physical embedding of OSAs on PLEO and the formation of complex coacervates between sodium alginate and chitosan. TG and DSC assay showed the chitosan/alginate/OSAs complex coacervates as wall materials substantially improved the thermal stability of PLEO. Besides, PLEO hydrogel beads had a better stability in aqueous and acidic food formulations, which achieved a complete and prolonged release of PLEO. The Peppas-Sahlin model was the best approach for PLEO release profile, and release phenomenon was mainly governed by Fickian diffusion.

Structural characterization and immunomodulatory activity of a polysaccharide from finger citron extracted by continuous phase-transition extraction.

FCP-2-1, a water-soluble polysaccharide rich in galacturonic acid was isolated by continuous phase-transition extraction and purified with DEAE-52 cellulose and Sephadex G-100 column chromatography from finger citron with essential oil and flavonoids removed. The structural characterization and immunomodulatory activity of FCP-2-1 were further investigated in this work. FCP-2-1 with a Mw and Mn of 1.503 × 104 g/mol and 1.125 × 104 g/mol, respectively, was predominantly composed of galacturonic acid, galactose, and arabinose in a molar ratio of 0.685: 0.032: 0.283. The main linkage types of FCP-2-1 were proved to be →5)-α-L-Araf-(1→ and →4)-α-D-GalpA-(1→ based on methylation and NMR analysis. Moreover, FCP-2-1 was demonstrated to have significant immunomodulatory effects on macrophages in vitro by improving the cell viability, and enhancing phagocytic activity and secretion of NO and cytokines (IL-1ß, IL-6, IL-10 and TNF-α), indicating that FCP-2-1 could be used as a natural agent in immunoregulation functional foods.

Investigation of bioactivity of unsaturated oligo­galacturonic acids produced from apple waste by Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 Pectinases.

Pectin is one of the main structural components in fruits and an indigestible fiber made of D-galacturonic acid units with α (1-4) linkage. This study investigates the microbial degradation of pectin in apple waste and the production of bioactive compounds. Firstly, pectin-degrading bacteria were isolated and identified, then pectinolytic activity was assessed by DNS. The products were evaluated by TLC and LC-MS-ESI. The antioxidative effects were investigated using DPPH and anti-cancer effects and cytotoxicity were analyzed by MTT and flow cytometry. In this study two new bacterial isolates, Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 with the pectinolytic enzyme were introduced. Structure analysis showed that the products of enzymatic degradation include unsaturated mono, di, tri, and penta galacturonic acids with 74% and 69% RSA at 40 mg/mL for A. faecalis and P. polymyxa S4, respectively. The results of anti-tumor properties on MCF-7 cells by MTT assay, for products of AGS3 and S4 at 40 mg/mL after 48 h, showed 7% and 9% survival, respectively. In the flow cytometric assessment, the compounds of AGS3 at 40 mg/mL were 100% lethal in 48 h and regarding S4 isolate caused 98% death. Cytotoxicity evaluation on L-929 cells showed no significant toxicity on living cells.

Hydrogel beads based on carboxymethyl cassava starch/alginate enriched with MgFe2O4 nanoparticles for controlling drug release.

Implementing novel oral drug delivery systems with controlled drug release behavior is valuable in cancer therapy. Herein, a green synthetic approach based on the sol-gel technique was adopted to prepare MgFe2O4 nanoparticles at different calcination temperatures using citric acid as a chelating/combustion agent. In this context, pH-responsive and magnetic carboxymethyl starch/alginate hydrogel beads (CMCS-SA) containing the MgFe2O4 nanoparticles were developed as potential drug carriers for the anticancer drug (Doxorubicin, Dox) release in simulated gastrointestinal fluids. Furthermore, in vitro release behaviors validated that these beads illustrated excellent stability in the simulated stomach liquids. In contrast, the data in simulated intestinal fluids showed sustained release of Dox because of their pH-sensitive swelling characteristics. Notably, applying an external magnetic field (EMF) could accelerate drug release from the beads. The in vitro release of drugs from gel beads was mainly accomplished by a combination of diffusion, swelling and erosion. Moreover, the cell cytotoxicity test and laser confocal results showed no harmful effects on normal cells (3T3) but were significant cytotoxic to colon cancer cell lines (HCT116) by drug-loaded hydrogel beads. Therefore, the prepared gel beads could be qualified as latent platforms for controlling the release of anticancer drugs in cancer treatment.

Evaluating Mannuronic Acid Effect on Gene Expression Profile of Inflammatory Mediators in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is a multisystem disorder. Various studies have shown the important role of inflammatory factors tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-22, MYD88, and toll-like receptor 2 (TLR2) in this disease. In this study, we investigated the anti-inflammatory effects of B-D-Mannuronic acid (M2000), as a new immunosuppressive drug, on the expression of these inflammatory markers in peripheral blood mononuclear cells (PBMCs) of RA patients. The blood samples of active RA patients and healthy volunteers were used for PBMCsl separation. The cells were cultured with LPS (1 µg/mL), low (5 µg/mL), moderate (25 µg/mL), and high (50 µg/mL) doses of M2000 and a single dose of diclofenac (1 µg/mL) to evaluate TNF-α, IL-6, IL-22, MYD88, and TLR2 genes expression by quantitative real-time (qRT-PCR). Cell surface expression and MFI of TLR2 were assessed; using flow cytometry. Our findings exhibited a significant reduction of TNF-α, IL-6, and MYD88 gene expressions after treatment with three doses of M2000 and an optimum dose of diclofenac. TLR2 gene expression was significantly diminished by moderate and high doses of M2000 and a single dose of diclofenac. Moreoversurface expression of TLR2 was significantly downregulated by moderate and high doses of M2000, while MFI of this receptor was significantly reduced by three doses of M2000. The results of this research showed that M2000 was able to significantly reduce the gene expression of inflammatory molecules  TNF-α, IL-6, MYD88, and TLR2 in patients PBMCs. factor-alpha; Rheumatoid arthritis. These data revealed a part of the molecular mechanisms of M2000 in the treatment process.

Structural Effects of pH Variation and Calcium Amount on the Microencapsulation of Glutathione in Alginate Polymers.

Reduced glutathione (GSH) has a high antioxidant capacity and is present in nearly every cell in the body, playing important roles in nutrient metabolism, antioxidant defense, and regulation of cellular events. Conversely, alginate is a macromolecule that has been widely used in the food, pharmaceutical, biomedical, and textile industries due to its biocompatibility, biodegradability, nontoxicity, and nonimmunogenicity as well as for its capabilities of retaining water and stabilizing emulsions. The primary goal of this study was to characterize and optimize the formation of a molecular complex of calcium alginate with GSH using a computational approach. As methods, we evaluated the influence of varying the amount of calcium cations at two different pHs on the structural stability of Ca2+-alginate complexes and thus on GSH liberation from these types of nanostructures. The results showed that complex stabilization depends on pH, with the system having a lower Ca2+ amount that produces the major GSH release. The systems at pH 2.5 retain more molecules within the calcium-alginate complex, which release GSH more slowly when embedded in more acidic media. In conclusions, this study demonstrates the dependence of the amount of calcium and the stabilizing effect of pH on the formation and subsequent maintenance of an alginate nanostructure. The results presented in this study can help to develop better methodological frameworks in industries where the release or capture of compounds, such as GSH in this case, depends on the conditions of the alginate nanoparticle.

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

The Evaluation of Safety Property and Apoptotic Efficacy of α-L-Guluronic Acid (G2013), as a Novel NSAID, Under In Vitro Examination on L929 and Hepatocellular Carcinoma Cell Lines.

BACKGROUND: Many investigations have expanded this concept that liver chronic inflammation has an essential role in persistent cell damages along with altering the liver microenvironment leading to fibrosis, cirrhosis, and finally, hepatocellular carcinoma (HCC). To reduce inflammation and relieve symptoms, Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are commonly used; however, their long-term usage can lead to severe adverse events on vital organs like the liver. Interestingly, the α-L-Guluronic Acid (G2013), as a novel NSAID with immunomodulatory properties, has shown the inhibitory effects on inflammation and metastasis in experimental models. OBJECTIVE: This study was conducted to determine the effects of G2013 on cytotoxicity and induction of apoptosis, as a new therapeutic target for cancer therapy, in the HepG2 cell line and the mouse fibroblast cell line L929, as a control. METHODS: MTT assay and flow cytometry method were carried out using the different concentrations of G2013 (5, 15, 25, 50, 100, 200 and 400 µg/ml) in 3 distinct incubation times. RESULTS: Our data showed that treatment of HepG2 cells with high concentration (400µg/mL) of G2013 could effectively cause a decrease in cell viability, so that they were statistically different after 72 hours compared to other concentrations (5 to 200 µg/ml) (p<0.05 and p<0.01, respectively). Moreover, the proportion of apoptosis of HepG2 cells at the dose of 200µg/mL considerably increased, suggesting that the induction of apoptosis by G2013 in HepG2 cells is dose- and time-dependent, which could promote its anticancer properties. CONCLUSION: The present study revealed that G2013 could induce apoptosis in the liver cancer model. Therefore, based on these findings, G2013 might be considered as a therapeutic option in cancer therapy.