3D culture of alginate-hyaluronic acid hydrogel supports the stemness of human mesenchymal stem cells.

The three-dimensional (3D) cell culture system is being employed more frequently to investigate cell engineering and tissue repair due to its close mimicry of in vivo microenvironments. In this study, we developed natural biomaterials, including hyaluronic acid, alginate, and gelatin, to mimic the creation of a 3D human mesenchymal stem cell (hMSC) extracellular environment and selected hydrogels with high proliferation capacity for 3D MSC culture. Human mesenchymal stem cells were encapsulated within hydrogels, and an investigation was conducted into the effects on cell viability and proliferation, stemness properties, and telomere activity compared to the 2D monolayer culture. Hydrogel characterization, cell proliferation, Live/Dead cell viability assay, gene expression, telomere relative length, and MSC stemness-related proteins by immunofluorescence staining were examined. The results showed that 3D alginate-hyaluronic acid (AL-HA) hydrogels increased cell proliferation, and the cells were grown as cellular spheroids within hydrogels and presented a high survival rate of 77.36% during the culture period of 14 days. Furthermore, the 3D alginate-hyaluronic acid (AL-HA) hydrogels increased the expression of stemness-related genes (OCT-4, NANOG, SOX2, and SIRT1), tissue growth and development genes (YAP and TAZ), and cell proliferation gene (Ki67) after culture for 14 days. Moreover, the telomere activity of the 3D MSCs was enhanced, as indicated by the upregulation of the human telomerase reverse transcriptase gene (hTERT) and the relative telomere length (T/S ratio) compared to the 2D monolayer culture. Altogether, these data suggest that the 3D alginate-hyaluronic acid (AL-HA) hydrogels could serve as a promising material for maintaining stem cell properties and might be a suitable carrier for tissue engineering proposals.

Comparison of the differentiation of ovine fetal bone-marrow mesenchymal stem cells towards osteocytes on chitosan/alginate/CuO-NPs and chitosan/alginate/FeO-NPs scaffolds.

In the current study, the creation of a chitosan/alginate scaffold hydrogel with and without FeO-NPs or CuO-NPs was studied. From fetal ovine bone marrow mesenchymal stem cells (BM-MSCs) were isolated and cultivated. Their differentiation into osteocyte and adipose cells was investigated. Also, on the scaffolds, cytotoxicity and apoptosis were studied. To investigate the differentiation, treatment groups include: (1) BM-MSCs were plated in DMEM culture medium with high glucose containing 10% FBS and antibiotics (negative control); (2) BM-MSCs were plated in osteogenic differentiation medium (positive control); (3) positive control group + FeO-NPs, (4) positive control group + CuO-NPs; (5) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate scaffold; (6) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate/FeO-NPs scaffold; and (7) BM-MSCs were plated in osteogenic differentiation medium on chitosan/alginate/CuO-NPs scaffold. Alkaline phosphatase enzyme concentrations, mineralization rate using a calcium kit, and mineralization measurement by alizarin staining quantification were evaluated after 21 days of culture. In addition, qRT-PCR was used to assess the expression of the ALP, ColA, and Runx2 genes. When compared to other treatment groups, the addition of CuO-NPs in the chitosan/alginate hydrogel significantly increased the expression of the ColA and Runx2 genes (p < 0.05). However, there was no significant difference between the chitosan/alginate hydrogel groups containing FeO-NPs and CuO-NPs in the expression of the ALP gene. It appears that the addition of nanoparticles, in particular CuO-NPs, has made the chitosan/alginate scaffold more effective in supporting osteocyte differentiation.

Chitosan-sodium alginate-polyethylene glycol-Ally isothiocyante nanocomposites ameliorates isoproterenol-induced myocardial infarction in rats.

Myocardial infarction (MI) is a common type of ischemic heart disease that affects millions of people worldwide. In recent times, nanotechnology has become a very promising field with immense applications. The current exploration was conducted to synthesize the chitosan-sodium alginate-polyethylene glycol-Ally isothiocyanate nanocomposites (CSP-AIso-NCs) and evaluate their beneficial roles against the isoproterenol (ISO)-induced MI in rats. The CSP-AIso-NCs were prepared and characterized by several characterization techniques. The MI was initiated in the rats by the administration of 85 mg/kg of ISO for 2 days and treated with 10 and 20 mg/kg of CSP-AIso-NCs for 1 month. The changes in heart weight and bodyweight were measured. The cardiac function markers were assessed with echocardiography. The lipid profiles, Na+, K+, and Ca2+ ions, cardiac biomarkers, antioxidant parameters, and inflammatory cytokines were assessed using corresponding assay kits. The histopathological study was done on the heart tissues. The UV spectral analysis revealed the maximum peak at 208 nm, which confirms the formation of CSP-AIso-NCs. The FT-IR analysis revealed the occurrence of different functional groups, and the crystallinity of the CSP-AIso-NCs was proved by the XRD analysis. DLS analysis indicated the size of the CSP-AIso-NCs at 146.50 nm. The CSP-AIso-NCs treatment increased the bodyweight and decreased the HW/BW ratio in the MI rats. The status of lipids was reduced, and HDL was elevated in the CSP-AIso-NCs administered to MI rats. CSP-AIso-NCs decreased the LVEDs, LVEDd, and NT-proBNP and increased the LVEF level. The oxidative stress markers were decreased, and the antioxidants were increased by the CSP-AIso-NCs treatment in the MI rats. The Na+ and Ca+ ions were reduced, and the K+ ions were increased by the CSP-AIso-NCs. The interleukin-1ß and tumor necrosis factor-α were also depleted, and Nrf-2 was improved in the CSP-AIso-NCs administered to MI rats. The histological study revealed the ameliorative effects of CSP-AIso-NCs. Overall, our outcomes revealed that the CSP-AIso-NCs are effective against the ISO-induced MI rats. Hence, it could be a hopeful therapeutic nanomedicine for MI treatment.

High-level production of a novel alginate lyase (FsAly7) from Flammeovirga sp. for efficient production of low viscosity soluble dietary fiber from sodium alginate.

Sodium alginate is one of the most abundant sustainable gum source for dietary fiber production. However, the preparation efficiencies of low viscosity soluble dietary fiber from sodium alginate remain low. Here, a novel alginate lyase gene (FsAly7) from Flammeovirga sp. was identified and high-level expressed in Pichia pastoris for low viscosity soluble dietary fiber production. The highest enzyme production of 3050 U mL-1 was achieved, which is by far the highest yield ever reported. FsAly7 was used for low viscosity soluble dietary fiber production from sodium alginate, and the highest degradation rate of 85.5 % was achieved under a high substrate content of 20 % (w/v). The molecular weight of obtained soluble dietary fiber converged to 10.75 kDa. FsAly7 catalyzed the cleavage of glycosidic bonds in alginate chains with formation of unsaturated non-reducing ends simultaneously in the degradation process, thus altered the chemical structures of hydrolysates. The soluble dietary fiber exhibited excellent properties, including low viscosity, high oil adsorption capacity activity (2.20 ± 0.03 g g-1) and high emulsifying activity (60.05 ± 2.96 mL/100 mL). This investigation may provide a novel alginate lyase catalyst as well as a solution for the efficient production of low viscosity soluble dietary fiber from sodium alginate.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

Extracellular DNA enhances biofilm integrity and mechanical properties of mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). P. aeruginosa becomes tolerant to antimicrobials in the biofilm state and is difficult to treat. Production of extracellular polymeric substances (EPS), such as alginate and extracellular DNA (eDNA), can allow adherence to abiotic and biotic surfaces, antimicrobial evasion, and resilience to environmental pressures. Alginate-producing mucoid variants of P. aeruginosa are frequently isolated from CF airway samples and are associated with worsening patient outcomes. While eDNA is a major structural component of nonmucoid P. aeruginosa biofilms, the potential role of eDNA in mucoid biofilms is unclear. Here, we investigate how eDNA contributes to clinical mucoid biofilm physiology and integrity. We predicted that eDNA plays a structural and mechanical role in mucoid biofilms. To test this, we quantified biofilm eDNA in mucoid biofilms and used microscopy and rheology to visualize eDNA and detect changes in biofilm structure and mechanics upon DNaseI treatment. We showed that biofilm eDNA abundance is diverse across clinical mucoid strains and observed a temporal increase in foci of eDNA within intact mucoid biofilms. Increased cell dispersal and reduced biomass were also observed following DNaseI treatment of mucoid biofilms. Degradation of eDNA also impacted the mechanical integrity of mucoid biofilms by increasing the stiffness and decreasing the cohesion of the biofilm. These findings advance our understanding of clinical mucoid P. aeruginosa biofilms and facilitate the development of new approaches to target biofilms by exploiting the functions of EPS components. IMPORTANCE Understanding the role of eDNA in mucoid Pseudomonas aeruginosa biofilms will lead to therapeutic strategies that combat the biophysical and structural function of EPS for the eradication of bacteria in mucoid biofilms during chronic infections. This knowledge can be used to further identify unknown matrix component interactions within pathogenic biofilm-forming clinical isolates.

Sodium alginate hydrogel integrated with type III collagen and mesenchymal stem cell to promote endometrium regeneration and fertility restoration.

A thinner endometrium has been linked to implantation failure, and various therapeutic strategies have been attempted to improve endometrial regeneration, including the use of mesenchymal stem cells (MSCs). However, low survival and retention rates of transplanted stem cells are main obstacles to efficient stem cell therapy in thin endometrium. Collagen type III is a key component of the extracellular matrix, plays a crucial role in promoting cell proliferation and differentiation, and has been identified as the major collagen expressed at the implantation site. Herein, composite alginate hydrogel containing recombinant type III collagen (rCo III) and umbilical cord mesenchymal stem cells are developed. rCo III serves as favorable bioactive molecule, displaying that rCo III administration promotes MSCs proliferation, stemness maintenance and migration. Moreover, rCo III administration enhances cell viability and migration of mouse endometrial stromal cells (ESCs). In a mouse model of thin endometrium, the Alg-rCo III hydrogel loaded with MSCs (MSC/Alg-rCo III) significantly induces endometrial regeneration and fertility enhancement in vivo. Further studies demonstrate that the MSC/Alg-rCo III hydrogel promoted endometrial function recovery partly by regulating mesenchymal-epithelial transition of ESCs. Taken together, the combination of Alg-rCo III hydrogel and MSCs has shown promising results in promoting endometrium regeneration and fertility restoration, and may provide new therapeutic options for endometrial disease.

Preparation and potential antitumor activity of alginate oligosaccharides degraded by alginate lyase from Cobetia marina.

It is of great significance to develop marine resources and study its potential biological activity by using alginate lyase produced by marine psychrophilic bacteria. In the previous study, a new marine psychrophilic bacterium (Cobetia marina HQZ08) was screened from the growth area of Laminaria japonica, and it was found that the strain could efficiently produce alginate-degrading enzyme (Aly30). In this paper, the ability of Aly30 to degrade alginate was optimized and the optimal degradation conditions were obtained. It was found that the main degradation product of alginate oligosaccharides was trisaccharide. In vitro cell experiments showed that the antitumor activity of low molecular weight alginate oligosaccharides was better than that of high molecular weight alginate oligosaccharides. In summary, Aly30 had the potential to produce alginate oligosaccharides with low degree of polymerization and antitumor activity, which provided a reference for the enzymatic preparation and application of alginate oligosaccharides.

Determining effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of clinical strains of Pseudomonas aeruginosa in biofilm-inspired alginate encapsulates.

BACKGROUND: Biofilms play a role in recalcitrance and treatability of bacterial infections, but majority of known antibiotic resistance mechanisms are biofilm-independent. Biofilms of Pseudomonas aeruginosa, especially in cystic fibrosis patients infected with the alginate producing strains in their lungs, are hard to treat. Changes in growth-related bacterial metabolism in biofilm affect their antibiotic recalcitrance which could be considered for new therapies designed based on these changes. In this study, effects of nitrate, arginine, and ferrous were investigated on antibiotic recalcitrance in alginate-encapsulated P. aeruginosa strains isolated from cystic fibrosis patients in the presence of amikacin, tobramycin, and ciprofloxacin. Also, expression of an efflux pump gene, mexY, was analyzed in selected strains in the presence of amikacin and ferrous. METHODS: Clinical P. aeruginosa strains were isolated from cystic fibrosis patients and minimum inhibitory concentration of amikacin, tobramycin, and ciprofloxacin was determined against all the strains. For each antibiotic, a susceptible and a resistant or an intermediate-resistant strain were selected, encapsulated into alginate beads, and subjected to minimal biofilm eradication concentration (MBEC) test. After determining MBECs, sub-MBEC concentrations (antibiotics at concentrations one level below the determined MBEC) for each antibiotic were selected and used to study the effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of encapsulated strains. Effects of ferrous and amikacin on expression of the efflux pump gene, mexY, was studied on amikacin sensitive and intermediate-resistant strains. One-way ANOVA and t test were used as the statistical tests. RESULTS: According to the results, the supplements had a dose-related effect on decreasing the number of viable cells; maximal effect was noted with ferrous, as ferrous supplementation significantly increased biofilm susceptibility to both ciprofloxacin and amikacin in all strains, and to tobramycin in a resistant strain. Also, treating an amikacin-intermediate strain with amikacin increased the expression of mexY gene, which has a role in P. aeruginosa antibiotic recalcitrance, while treating the same strain with ferrous and amikacin significantly decreased the expression of mexY gene, which was a promising result. CONCLUSIONS: Our results support the possibility of using ferrous and arginine as an adjuvant to enhance the efficacy of conventional antimicrobial therapy of P. aeruginosa infections.

Collagen-alginate 3D microscaffolds for studying cellular migration.

Metastasis is one of the major causes for cancer mortality. Its early steps comprise of invasion of basement membrane and migration. Thus, it is hypothesized that a platform, that allows quantification and grading of migration capability of cells can potentially be used for predicting metastatic potential. Two-dimensional (2D) models have been rendered inadequate for modelling in-vivo microenvironment due to various reasons. To attenuate homogeneity observed in 2D, three-dimensional (3D) platforms supplemented with bioinspired components have been designed. Unfortunately, till date there are no simple models to capture the migration of cells in 3D along with quantification of the process. In this study, we report an alginate-collagen based 3D model system, which can predict the migratory property of the cells within 72 h. The micron size of the scaffold enabled faster readout and the optimum pore-size provided conducive cellular growth environment. The platform's ability to allow observation of cellular migration was validated by encapsulating cells with transiently upregulated matrix metalloprotease 9 (MMP9), which has been reported to play a significant role in migration of cells during metastasis. The readout for migration was clustering of cells in the microscaffolds detected in a short span of 48 h. The observed clustering in MMP9 upregulated cells was validated by observing changes in the epithelial-mesenchymal transition (EMT) markers. Thus, this simple 3D platform can be used to study migration and predict the metastatic potential of cells.

How Three Self-Secreted Biofilm Exopolysaccharides of Pseudomonas aeruginosa, Psl, Pel, and Alginate, Can Each Be Exploited for Antibiotic Adjuvant Effects in Cystic Fibrosis Lung Infection.

In cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a cause of increased morbidity and mortality, especially in patients for whom infection becomes chronic and there is reliance on long-term suppressive therapies. Current antimicrobials, though varied mechanistically and by mode of delivery, are inadequate not only due to their failure to eradicate infection but also because they do not halt the progression of lung function decline over time. One of the reasons for this failure is thought to be the biofilm mode of growth of P. aeruginosa, wherein self-secreted exopolysaccharides (EPSs) provide physical protection against antibiotics and an array of niches with resulting metabolic and phenotypic heterogeneity. The three biofilm-associated EPSs secreted by P. aeruginosa (alginate, Psl, and Pel) are each under investigation and are being exploited in ways that potentiate antibiotics. In this review, we describe the development and structure of P. aeruginosa biofilms before examining each EPS as a potential therapeutic target for combating pulmonary infection with P. aeruginosa in CF, with a particular focus on the current evidence for these emerging therapies and barriers to bringing these therapies into clinic.

Alginate sulfate/ECM composite hydrogel containing electrospun nanofiber with encapsulated human adipose-derived stem cells for cartilage tissue engineering.

Stem cell therapy is a promising strategy for cartilage tissue engineering, and cell transplantation using polymeric scaffolds has recently gained attention. Herein, we encapsulated human adipose-derived stem cells (hASCs) within the alginate sulfate hydrogel and then added them to polycaprolactone/gelatin electrospun nanofibers and extracellular matrix (ECM) powders to mimic the cartilage structure and characteristic. The composite hydrogel scaffolds were developed to evaluate the relevant factors and conditions in mechanical properties, cell proliferation, and differentiation to enhance cartilage regeneration. For this purpose, different concentrations (1-5 % w/v) of ECM powder were initially loaded within an alginate sulfate solution to optimize the best composition for encapsulated hASCs viability. Adding 4 % w/v of ECM resulted in optimal mechanical and rheological properties and better cell viability. In the next step, electrospun nanofibrous layers were added to the alginate sulfate/ECM composite to prepare different layered hydrogel-nanofiber (2, 3, and 5-layer) structures with the ability to mimic the cartilage structure and function. The 3-layer structure was selected as the optimum layered composite scaffold, considering cell viability, mechanical properties, swelling, and biodegradation behavior; moreover, the chondrogenesis potential was assessed, and the results showed promising features for cartilage tissue engineering application.

Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Process and applications of alginate oligosaccharides with emphasis on health beneficial perspectives.

Alginates are linear polymers comprising 40% of the dry weight of algae possess various applications in food and biomedical industries. Alginate oligosaccharides (AOS), a degradation product of alginate, is now gaining much attention for their beneficial role in food, pharmaceutical and agricultural industries. Hence this review was aimed to compile the information on alginate and AOS (prepared from seaweeds) during 1994-2020. As per our knowledge, this is the first review on the potential use of alginate oligosaccharides in different fields. The alginate derivatives are grouped according to their applications. They are involved in the isolation process and show antimicrobial, antioxidant, anti-inflammatory, antihypertension, anticancer, and immunostimulatory properties. AOS also have significant applications in prebiotics, nutritional supplements, plant growth development and others products.

Distinct Long- and Short-Term Adaptive Mechanisms in Pseudomonas aeruginosa.

Heterogeneous environments such as the chronically infected cystic fibrosis lung drive the diversification of Pseudomonas aeruginosa populations into, e.g., mucoid, alginate-overproducing isolates or small-colony variants (SCVs). In this study, we performed extensive genome and transcriptome profiling on a clinical SCV isolate that exhibited high cyclic diguanylate (c-di-GMP) levels and a mucoid phenotype. We observed a delayed, stepwise decrease of the high levels of c-di-GMP as well as alginate gene expression upon passaging the SCV under noninducing, rich medium growth conditions over 7 days. Upon prolonged passaging, this lagging reduction of the high c-di-GMP levels under noninducing planktonic conditions (reminiscent of a hysteretic response) was followed by a phenotypic switch to a large-colony morphology, which could be linked to mutations in the Gac/Rsm signaling pathway. Complementation of the Gac/Rsm signaling-negative large-colony variants with a functional GacSA system restored the SCV colony morphotype but was not able to restore the high c-di-GMP levels of the SCV. Our data thus suggest that expression of the SCV colony morphotype and modulation of c-di-GMP levels are genetically separable and follow different evolutionary paths. The delayed switching of c-di-GMP levels in response to fluctuating environmental conditions might provide a unique opportunity to include a time dimension to close the gap between short-term phenotypic and long-term genetic adaptation to biofilm-associated growth conditions. IMPORTANCE Extreme environments, such as those encountered during an infection process in the human host, make effective bacterial adaptation inevitable. While bacteria adapt individually by activating stress responses, long-term adaptation of bacterial communities to challenging conditions can be achieved via genetic fixation of favorable traits. In this study, we describe a two-pronged bacterial stress resistance strategy in the opportunistic pathogen Pseudomonas aeruginosa. We show that the production of adjusted elevated c-di-GMP levels, which drive protected biofilm-associated phenotypes in vivo, resembles a stable hysteretic response which prevents unwanted frequent switching. Cellular hysteresis might provide a link between individual adaptability and evolutionary adaptation to ensure the evolutionary persistence of host-adapted stress response strategies.

Alginate Alleviates Dextran Sulfate Sodium-Induced Colitis by Promoting Bifidobacterium animalis and Intestinal Hyodeoxycholic Acid Synthesis in Mice.

Alginate (ALG) is known to alleviate intestinal inflammation in inflammatory bowel disease, but its mechanism of action remains elusive. In the present study, we studied the involvement of the intestinal microbiota and bile acid (BA) metabolism in ALG-mediated anti-inflammatory effects in mice. A combination of 16S rRNA gene amplicon sequencing, shotgun metagenomic sequencing, and targeted BA metabolomic profiling was employed to investigate structural and functional differences in the colonic microbiota and BA metabolism in dextran sulfate sodium (DSS)-treated mice with or without dietary supplementation of ALG. We further explored the role of the intestinal microbiota as well as a selected ALG-enriched bacterium and BA in DSS-induced colitis. Dietary ALG alleviated DSS-mediated intestinal inflammation and enriched a small set of bacteria including Bifidobacterium animalis in the colon (P < 0.05). Additionally, ALG restored several bacteria carrying secondary BA-synthesizing enzymes such as 7α-hydroxysteroid dehydrogenase and BA hydrolase to healthy levels in DSS-treated mice. Although a majority of BAs were suppressed by DSS, a few secondary BAs such as hyodeoxycholic acid (HDCA) were markedly enriched by ALG. Furthermore, ALG significantly upregulated the expression of a major BA receptor, the farnesoid X receptor, while suppressing NF-κB and c-Jun N-terminal kinase (JNK) activation. Depletion of the intestinal microbiota completely abrogated the protective effect of ALG in DSS-treated mice. Similar to ALG, B. animalis and HDCA exerted a strong anti-inflammatory effect in DSS-induced colitis by downregulating inflammatory cytokines (interleukin-1ß [IL-1ß], IL-6, and tumor necrosis factor alpha [TNF-α]). Taken together, these results indicated that ALG achieves its alleviating effect on intestinal inflammation through regulation of the microbiota by enriching B. animalis to promote the biosynthesis of specific secondary BAs such as HDCA. These findings have revealed intricate interactions among the intestinal microbiota, BA metabolism, and intestinal health and further provided a novel strategy to improve intestinal health through targeted manipulation of the intestinal microbiota and BA metabolism. IMPORTANCE ALG has been shown to ameliorate inflammatory bowel disease (IBD), but little is known about the mechanism of its anti-inflammatory action. This study was the first to demonstrate that ALG provided a preventive effect against colitis in an intestinal microbiota-dependent manner. Furthermore, we confirmed that by selectively enriching intestinal B. animalis and secondary BA (HDCA), ALG contributed to the attenuation of DSS-induced colitis. These findings contribute to a better understanding of the mechanism of action of ALG on the attenuation of colitis and provide new approaches to IBD therapy by regulating gut microbial BA metabolism.

Construction of Magnetic Composite Bacterial Carrier and Application in 17ß-Estradiol Degradation.

Estrogen contamination is widespread and microbial degradation is a promising removal method; however, unfavorable environments can hinder microbial function. In this study, a natural estrogen 17ß-estradiol (E2) was introduced as a degradation target, and a new combination of bacterial carrier was investigated. We found the best combination of polyvinyl alcohol (PVA) and sodium alginate (SA) was 4% total concentration, PVA:SA = 5:5, with nano-Fe3O4 at 2%, and maltose and glycine added to promote degradation, for which the optimal concentrations were 5 g·L-1 and 10 g·L-1, respectively. Based on the above exploration, the bacterial carrier was made, and the degradation efficiency of the immobilized bacteria reached 92.3% in 5 days. The immobilized bacteria were reused for three cycles, and the degradation efficiency of each round could exceed 94%. Immobilization showed advantages at pH 5, pH 11, 10 °C, 40 °C, and 40 g·L-1 NaCl, and the degradation efficiency of the immobilized bacteria was higher than 90%. In the wastewater, the immobilized bacteria could degrade E2 to about 1 mg·L-1 on the 5th day. This study constructed a bacterial immobilization carrier using a new combination, explored the application potential of the carrier, and provided a new choice of bacterial immobilization carrier.

Development of non-adherent cell-enclosing domes with enzymatically cross-linked hydrogel shell.

Non-adherent cells, such as hematopoietic cells and lymphocytes, are important research subjects in medical and biological fields. Therefore, a system that enables the handling of non-adherent cells in solutions in the same manner as that of adhering cells during medium exchange, exposure to chemicals, washing, and staining in imaging applications would be useful. Here, we report a 'Cell Dome' platform in which non-adherent cells can be enclosed and grown in the cavities of about 1 mm diameter and 270µm height. The domes consist of an alginate-based hydrogel shell of 90µm thickness. Cell Domes were formed on glass plates by horseradish peroxidase-mediated cross-linking. Human leukaemia cell line K562 cells enclosed in Cell Domes were stable for 29 days with every 2-3 days of medium change. The enclosed cells grew in the cavities and were stained and differentiated with reagents supplied from the surrounding medium. Additionally, K562 cells that filled the cavities (a 3D microenvironment) were more hypoxic and highly resistant to mitomycin C than those cultured in 2D. These findings demonstrate that the 'Cell Dome' may be a promising tool for conveniently culturing and evaluating non-adherent cells.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization.

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 µM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 µM.