Polysaccharide Matrix Research Articles

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Synergistic interaction of hTGF-\u03b23 with hBMP-6 promotes articular cartilage formation in chitosan scaffolds with hADSCs: implications for regenerative medicine

BackgroundHuman TGF-β3 has been used in many studies to induce genes coding for typical cartilage matrix components and accelerate chondrogenic differentiation, making it the standard constituent in most cultivation media used for the assessment of chondrogenesis associated with various stem cell types on carrier matrices. However, in vivo data suggests that TGF-β3 and its other isoforms also induce endochondral and intramembranous osteogenesis in non-primate species to other mammals. Based on previously demonstrated improved articular cartilage induction by a using hTGF-β3 and hBMP-6 together on hADSC cultures and the interaction of TGF- β with matrix in vivo, the present study investigates the interaction of a chitosan scaffold as polyanionic polysaccharide with both growth factors. The study analyzes the difference between chondrogenic differentiation that leads to stable hyaline cartilage and the endochondral ossification route that ends in hypertrophy by extending the usual panel of investigated gene expression and stringent employment of quantitative PCR.ResultsBy assessing the viability, proliferation, matrix formation and gene expression patterns it is shown that hTGF-β3 + hBMP-6 promotes improved hyaline articular cartilage formation in a chitosan scaffold in which ACAN with Col2A1 and not Col1A1 nor Col10A1 where highly expressed both at a transcriptional and translational level. Inversely, hTGF-β3 alone tended towards endochondral bone formation showing according protein and gene expression patterns.ConclusionThese findings demonstrate that clinical therapies should consider using hTGF-β3 + hBMP-6 in articular cartilage regeneration therapies as the synergistic interaction of these morphogens seems to ensure and maintain proper hyaline articular cartilage matrix formation counteracting degeneration to fibrous tissue or ossification. These effects are produced by interaction of the growth factors with the polysaccharide matrix.

Living the Sweet Life: How Liquorilactobacillus hordei TMW 1.1822 Changes Its Behavior in the Presence of Sucrose in Comparison to Glucose.

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests that L. hordei TMW 1.1822 favors fructose over other sugars. The dextransucrase was expressed irrespectively of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix but is also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for its use as a starter culture in plant-based food fermentations with in situ dextran formation.

Abstract 322: A unique ex vivo tumor model: 3D co-cultured system with cancer cells and stromal tissue for drug evaluation

Abstract Background;Recently, three-dimensional culture model has been reported as better tumor model than 2D culture. The importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and resistance against treatment. So that ex vivo model which recaptures the tumor microenvironment is required to perform drug evaluation mimicking the patient tumor tissue. Methods; Layered 3D stromal tissues with microvascular network were produced by culturing normal fibroblasts and endothelial cells coated with the extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and co-cultured tumor were morphologically evaluated by HE staining, immunohistochemistry and immunofluorescence (IF). Drug sensitivity assay were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in JFCR under approved IRB. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Furthermore, in vivo drug sensitivity was also evaluated. Results: The CD31 positive luminal structures which shows vascular endothelial tube formation were observed intra multi-layered tissues. The dense microvascular network tended to be formed nearby cancer cells. In comparison with 2D culture model, decreased drug sensitivities were represented in the layered 3D co-cultured model. The evaluated drug sensitivities in 3D model may reflect the response of cancer cells in in vivo. Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Our unique 3D ex vivo model appear to be a valuable tool for drug evaluation, and thus further studies are underway to confirm the usefulness of our model. Testing approved and/or developing compounds with PDCs in the model would potentially enable better prediction their efficacy. Citation Format: Yuki Takahashi, Kei Tsukamoto, Yuki Shimizu, Ryohei Katayama, Eiji Shinozaki, Satoshi Nagayama, Michiya Matsusaki, Shiro Kitano, Kensei Yamaguchi, Naoya Fujita. A unique ex vivo tumor model: 3D co-cultured system with cancer cells and stromal tissue for drug evaluation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 322.

Abstract 3877: A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into microenviroment

Abstract Background The modulation of the immune system is a proven key target for cancer therapy, however, the complex interplay between immune cells and cancer cells in cancer microenvironment remains only partially understood. In addition, the currently available in vitro cancer microenvironment models are not designed to study the immunologic agents and a role of immune cells. Here, we developed a unique layered 3D stromal tissue model comprising immune, stromal and cancer cells. Method Layered 3D stromal tissue model was constructed by culturing fibroblasts and endothelial cells coated with extracellular matrix (ECM) and natural polysaccharide. Morphological analysis of the layered 3D stromal tissues and co-cultured tumor were evaluated by HE stain, immunohistochemistry and immunofluorescence (IF). For cytotoxicity assay, cancer cell lines were seeded on or in the 3D stromal tissue and incubated with peripheral blood mononuclear cell (PBMC) and Programmed cell death 1 (PD-1) blockade with Nivolumab. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Result The co-cultured stromal tissues were multi-layered (approximately 20 layers), had microvascular formation. Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) derived from fibroblast cells played important roles in tube formation. Moreover, the position of cancer cells in 3D stromal tissue was manipulated easily. PBMC infiltration into 3D stromal tissue and the interaction between PBMC and cancer cells were visualized. High level of programmed cell death ligand 1 (PD-L1) expression on cancer cells correlated with a higher response to PD-1 blockade in vitro as well as clinical trials. Conclusion We present a unique layered 3D cell culture system based on stromal/cancer/immune cells to study immunologic agents and immune cell infiltration in cancer microenvironment. Our 3D model has the ability to reflect in vivo conditions closer than 2D model and provide a tool for in vitro tumor infiltrating lymphocyte (TIL) or immunologic agent testing. Citation Format: Rii Morimura, Isana Nada, Kei Tsukamoto, Eiji Shinozaki, shiro kitano. A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into microenviroment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3877.

The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.

Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement

BackgroundReconstruction of metaphyseal fractures represents a clinical challenge for orthopedic surgeons. Especially in osteoporotic bone, these fractures are frequently accompanied by osseous substance defects. In order to ensure rapid mobilization of patients, high stability requirements must be met by osteosynthesis. Various bone graft materials have been introduced in the past, such as autologous bone or exogenous bone substitute materials. These are used as bone void fillers or as augmentation techniques to ensure safe fixation of osteosynthesis. New calcium phosphate-based bone void-filling materials could be a promising alternative to autologous bone or to the currently and widely used polymethylmethacrylate (PMMA)-based cement. The aim of this study was to evaluate a novel paste-like bone void filler in vivo and in vitro with regard to biocompatibility and osteoconductivity.MethodsIn addition to in vitro testing of cell compatibility using pre-osteoblasts (MC3T3-E1), 35 Wistar rats were treated in vivo with implantation of various material mixtures based on calcium phosphate and aluminum oxide reinforcement in a metaphyseal drill hole defect. After 4 weeks, an examination by micro-computed tomography (μCT) and histology was performed.ResultsThe in vitro analysis showed good biocompatibility with a high cell survival of osteoblasts. In the in vivo experiments, a significantly higher bone ingrowth compared to the empty defect was shown by μCT and histological analysis. Here, the group receiving material reinforced with aluminum oxide (Al2O3) showed a bone volume/tissue volume (BV/TV) of 89.19% compared to a BV/TV of 83.14% for the empty defect (p = 0.0013). In the group treated with a polysaccharide matrix, no increase in BV/TV was observed given a mean ratio of 80.14%. Scoring of histological sections did not reveal a significant difference between CaP and CaP that was substituted with Al2O3.ConclusionThe results of this study show an encouraging first step towards the development of new pasty, bone void-filling materials. We demonstrated that a new paste-like bone-filling material, based on calcium phosphate granulates and aluminum oxide to provide strength, exhibits good biocompatibility and osteoconductivity. Further biomechanical test in an osteoporotic animal model will have to be performed, to prove feasibility in metaphyseal defects.

The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively.

Quorum sensing (QS) is often required for the formation of bacterial biofilms and is a popular target of biofilm control strategies. Previous studies implicate the ComQXPA quorum sensing system of Bacillus subtilis as a promoter of biofilm formation. Here, we report that ComX signaling peptide deficient mutants form thicker and more robust pellicle biofilms that contain chains of cells. We confirm that ComX positively affects the transcriptional activity of the PepsA promoter, which controls the synthesis of the major matrix polysaccharide. In contrast, ComX negatively controls the PtapA promoter, which drives the production of TasA, a fibrous matrix protein. Overall, the biomass of the mutant biofilm lacking ComX accumulates more monosaccharide and protein content than the wild type. We conclude that this QS phenotype might be due to extended investment into growth rather than spore development. Consistent with this, the ComX deficient mutant shows a delayed activation of the pre-spore specific promoter, PspoIIQ, and a delayed, more synchronous commitment to sporulation. We conclude that ComX mediated early commitment to sporulation of the wild type slows down biofilm formation and modulates the coexistence of multiple biological states during the early stages of biofilm development.

The effect of alginate composition on adsorption to calcium carbonate surfaces

Bacterial anchoring to limestone rocks is thought to occur by selective adsorption of biomolecules found in the extracellular matrix, such as polysaccharides. Here we study the adsorbed structure of a model matrix polysaccharide, sodium alginate, at the calcite/water interface using neutron reflection (NR). Sodium alginate was found to form highly hydrated layers extending up to 350 Å into solution at concentrations up to 2.5 ppm (the inflection point of the adsorption isotherm). The adsorption of alginate was driven by dissolution of the calcite surface through complexation of free calcium ions. This was shown using two alginates with differing ratios of sugar residues. Alginates with a higher proportion of guluronic acid (G) have a higher affinity for calcium ions and were found to cause the surface to dissolve to a greater extent and to adsorb more at the surface when compared to alginates with a higher proportion of mannuronic acid (M). Adding magnesium to the high G alginate solution reduced dissolution of the surface and the adsorbed amount. In this work, we have shown that polysaccharide adsorption to sparingly soluble calcite interfaces is closely related to polymer conformation and affinity for free calcium ions in solution.

Hydroxypropyl methylcellulose hydrogel of berberine chloride-loaded escinosomes: Dermal absorption and biocompatibility

Aim of this work was to prepare and characterize new nanocarrier-loaded hydrogel formulations for topical application, using hydroxypropyl methylcellulose (HMPC) and special nanovesicles, the escinosomes. The combination of the two technological strategies, nanocarriers and hydrogels, was selected to circumvent some drawbacks of nanovesicles and develop stable and efficient skin-delivery platforms. HPMC is a derivative of cellulose with a wide range of physicochemical properties, forming suitable hydrogel for dermatological applications. Escinosomes, made of escin (ESN), a natural bioactive saponin, plus phosphatidylcholine, were loaded with berberine chloride (BRB), a bioactive natural product, and were entrapped in the polymeric matrix of HPMC. Release and permeation properties of aqueous ESN and BRB dispersions, escinosomes were compared with the corresponding hydrogels. Viscosity measurements evidenced their suitability for topical applications. In vitro permeation experiments showed a higher residence time of the HPMC-hydrogel. Thus, the new escinosome HPMC-hydrogel formulations combine the advantages of a modified release and increased transdermal permeability (escinosome components), with better viscosity properties (polysaccharide matrix). In addition, the developed HPMC-hydrogels also had a very good safety profile and skin biocompatibility studies showed no potentially hazardous skin irritation. Finally, the developed escinosome HMPC-hydrogel formulations were very stable with appropriate mechanical properties.

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

Positive Effect of Cold Atmospheric Nitrogen Plasma on the Behavior of Mesenchymal Stem Cells Cultured on a Bone Scaffold Containing Iron Oxide-Loaded Silica Nanoparticles Catalyst.

Low-temperature atmospheric pressure plasma was demonstrated to have an ability to generate different reactive oxygen and nitrogen species (RONS), showing wide biological actions. Within this study, mesoporous silica nanoparticles (NPs) and FexOy/NPs catalysts were produced and embedded in the polysaccharide matrix of chitosan/curdlan/hydroxyapatite biomaterial. Then, basic physicochemical and structural characterization of the NPs and biomaterials was performed. The primary aim of this work was to evaluate the impact of the combined action of cold nitrogen plasma and the materials produced on proliferation and osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (ADSCs), which were seeded onto the bone scaffolds containing NPs or FexOy/NPs catalysts. Incorporation of catalysts into the structure of the biomaterial was expected to enhance the formation of plasma-induced RONS, thereby improving stem cell behavior. The results obtained clearly demonstrated that short-time (16s) exposure of ADSCs to nitrogen plasma accelerated proliferation of cells grown on the biomaterial containing FexOy/NPs catalysts and increased osteocalcin production by the cells cultured on the scaffold containing pure NPs. Plasma activation of FexOy/NPs-loaded biomaterial resulted in the formation of appropriate amounts of oxygen-based reactive species that had positive impact on stem cell proliferation and at the same time did not negatively affect their osteogenic differentiation. Therefore, plasma-activated FexOy/NPs-loaded biomaterial is characterized by improved biocompatibility and has great clinical potential to be used in regenerative medicine applications to improve bone healing process.

Tailoring Nanoparticle-Biofilm Interactions to Increase the Efficacy of Antimicrobial Agents Against Staphylococcus aureus.

BackgroundConsidering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing old drugs and improving antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections but produce different biofilm matrices. MSSA biofilm cells are typically embedded in an extracellular polysaccharide matrix, whereas MRSA biofilms comprise predominantly of surface proteins and extracellular DNA (eDNA). Nanoparticles (NPs) have the potential to enhance the delivery of antimicrobial agents into biofilms. However, the mechanisms which influence the interactions between NPs and the biofilm matrix are not yet fully understood.MethodsTo investigate the influence of NPs surface chemistry on vancomycin (VAN) encapsulation and NP entrapment in MRSA and MSSA biofilms, mesoporous silica nanoparticles (MSNs) with different surface functionalization (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised using an adapted Stöber method. The antibacterial efficacy of VAN-loaded MSNs was assessed against MRSA and MSSA biofilms.ResultsThe two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL−1) correlated with the reduced viability of both MSSA and MRSA biofilm cells. This allowed the administration of low MSNs concentrations while maintaining a high local concentration of the antibiotic surrounding the bacterial cells.ConclusionOur data suggest that by tailoring the surface functionalization of MSNs, enhanced bacterial cell targeting can be achieved, leading to a novel treatment strategy for biofilm infections.

Influence of an Orange Product Composition on the Characteristics of the Obtained Freeze-dried Cake and Powder as Related to Their Consumption Pattern

Freeze-drying is a good alternative to obtain high-quality fruit products. Process variables but also product composition may affect the properties of the obtained food. In this study, the water and solute composition of an orange product was varied by using the puree or the juice, by adding gum Arabic and bamboo fiber as carriers and by heating the mix or adding water. Water, °Brix, vitamin C, total phenol, and carotenoid content were quantified. The porosity and mechanical properties of the freeze-dried cake also as the particle size distribution, angle of repose, wetting time of the powder, and viscosity when rehydrated were analyzed. The greater amount of insoluble solute in the puree leads to a more porous and crunchier cake with higher carotenoid content. The presence of more soluble in the juice, vitamin C included, favors the powder flowability and wettability, the rehydrated product being less viscous. Gum Arabic with enough water forms a polysaccharide matrix that partially retains the vitamin C, leads to a more fragile freeze-dried cake, and provides a rehydrated powder lower in viscosity. Heating the mix negatively affects the carotenoids while favors vitamin C extraction and promotes a gelled pectin matrix that markedly extends the wetting time of the powder and increases the viscosity when rehydrated. To sum up, the orange puree is recommended for the purposes of obtaining a crunchy snack-type product and the juice when a powder to be rehydrated is desired, in this case avoiding any heat treatment before freeze-drying.

FRA1 Kinesin Modulates the Lateral Stability of Cortical Microtubules through Cellulose Synthase-Microtubule Uncoupling Proteins.

Cell wall assembly requires harmonized deposition of cellulose and matrix polysaccharides. Cortical microtubules orient the deposition of cellulose by guiding the trajectory of cellulose synthase complexes. Vesicles containing matrix polysaccharides are thought to be transported by the FRAGILE FIBER1 (FRA1) kinesin to facilitate their secretion along cortical microtubules. The cortical microtubule cytoskeleton thus may provide a platform to coordinate the delivery of cellulose and matrix polysaccharides, but the underlying molecular mechanisms remain unknown. Here, we show that the tail region of the Arabidopsis (Arabidopsis thaliana) FRA1 kinesin physically interacts with cellulose synthase-microtubule uncoupling (CMU) proteins that are important for the microtubule-dependent guidance of cellulose synthase complexes. Interaction with CMUs did not affect microtubule binding or motility of the FRA1 kinesin but differentially affected the protein levels and microtubule localization of CMU1 and CMU2, thus regulating the lateral stability of cortical microtubules. Phosphorylation of the FRA1 tail region inhibited binding to CMUs and consequently reversed the extent of cortical microtubule decoration by CMU1 and CMU2. Genetic experiments demonstrated the significance of this interaction to the growth and reproduction of Arabidopsis plants. We propose that modulation of CMU protein levels and microtubule localization by FRA1 provides a mechanism that stabilizes the sites of deposition of both cellulose and matrix polysaccharides.

Marine Protists and Rhodotorula Yeast as Bio-Convertors of Marine Waste into Nutrient-Rich Deposits for Mangrove Ecosystems

This paper represents a comprehensive study of two new thraustochytrids and a marine Rhodotorula red yeast isolated from Australian coastal waters for their abilities to be a potential renewable feedstock for the nutraceutical, food, fishery and bioenergy industries. Mixotrophic growth of these species was assessed in the presence of different carbon sources: glycerol, glucose, fructose, galactose, xylose, and sucrose, starch, cellulose, malt extract, and potato peels. Up to 14g DW/L (4.6gDW/L-day and 2.8gDW/L-day) of biomass were produced by Aurantiochytrium and Thraustochytrium species, respectively. Thraustochytrids biomass contained up to 33% DW of lipids, rich in omega-3 polyunsaturated docosahexaenoic acid (C22:6, 124mg/g DW); up to 10.2mg/gDW of squalene and up to 61μg/gDW of total carotenoids, composed of astaxanthin, canthaxanthin, echinenone, and β-carotene. Along with the accumulation of these added-value chemicals in biomass, thraustochytrid representatives showed the ability to secrete extracellular polysaccharide matrixes containing lipids and proteins. Rhodotorula sp lipids (26% DW) were enriched in palmitic acid (C16:0, 18mg/gDW) and oleic acid (C18:1, 41mg/gDW). Carotenoids (87μg/gDW) were mainly represented by β-carotene (up to 54μg/gDW). Efficient growth on organic and inorganic sources of carbon and nitrogen from natural and anthropogenic wastewater pollutants along with intracellular and extracellular production of valuable nutrients makes the production of valuable chemicals from isolated species economical and sustainable.

The exopolysaccharide\u2013eDNA interaction modulates 3D architecture of Bacillus subtilis biofilm

BackgroundBacterial biofilms are surface-adherent microbial communities in which individual cells are surrounded by a self-produced extracellular matrix of polysaccharides, extracellular DNA (eDNA) and proteins. Interactions among matrix components within biofilms are responsible for creating an adaptable structure during biofilm development. However, it is unclear how the interactions among matrix components contribute to the construction of the three-dimensional (3D) biofilm architecture.ResultsDNase I treatment significantly inhibited Bacillus subtilis biofilm formation in the early phases of biofilm development. Confocal laser scanning microscopy (CLSM) and image analysis revealed that eDNA was cooperative with exopolysaccharide (EPS) in the early stages of B. subtilis biofilm development, while EPS played a major structural role in the later stages. In addition, deletion of the EPS production gene epsG in B. subtilis SBE1 resulted in loss of the interaction between EPS and eDNA and reduced the biofilm biomass in pellicles at the air-liquid interface. The physical interaction between these two essential biofilm matrix components was confirmed by isothermal titration calorimetry (ITC).ConclusionsBiofilm 3D structures become interconnected through surrounding eDNA and EPS. eDNA interacts with EPS in the early phases of biofilm development, while EPS mainly participates in the maturation of biofilms. The findings of this study provide a better understanding of the role of the interaction between eDNA and EPS in shaping the biofilm 3D matrix structure and biofilm formation.

Pectic galactan affects cell wall architecture during secondary cell wall deposition.

β-(1,4)-galactan determines the interactions between different matrix polysaccharides and cellulose during the cessation of cell elongation. Despite recent advances regarding the role of pectic β-(1,4)-galactan neutral side chains in primary cell wall remodelling during growth and cell elongation, little is known about the specific function of this polymer in other developmental processes. We have used transgenic Arabidopsis plants overproducing chickpea βI-Gal β-galactosidase under the 35S CaMV promoter (35S::βI-Gal) with reduced galactan levels in the basal non-elongating floral stem internodes to gain insight into the role of β-(1,4)-galactan in cell wall architecture during the cessation of elongation and the beginning of secondary growth. The loss of galactan mediated by βI-Gal in 35S::βI-Gal plants is accompanied by a reduction in the levels of KOH-extracted xyloglucan and an increase in the levels of xyloglucan released by a cellulose-specific endoglucanase. These variations in cellulose-xyloglucan interactions cause an altered xylan and mannan deposition in the cell wall that in turn results in a deficient lignin deposition. Considering these results, we can state that β-(1,4)-galactan plays a key structural role in the correct organization of the different domains of the cell wall during the cessation of growth and the early events of secondary cell wall development. These findings reinforce the notion that there is a mutual dependence between the different polysaccharides and lignin polymers to form an organized and functional cell wall.

Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides

Multicellular spheroids (spheroids) are expected to be a promising approach to mimic in vivo organ functions and cell microenvironments. However, conventional spheroids do not fully consider the existence of extracellular matrices (ECMs). In this study, we developed a tunable method for replenishing macromolecules, including ECM components and polysaccharides, into spheroids without compromising cell viability by injecting a microvolume cell suspension into a high density of methylcellulose dissolved in the culture medium. Adjusting the ECM concentration in the cell suspension enabled the generation of different three-dimensional microstructures, such as “ECM gel capsules”, which contained individually separated cells, and “ECM-loaded spheroids”, which had thin ECM layers between cells. ECM-loaded spheroids with a 30-fold dilution of Matrigel (0.3 mg/ml) showed significantly higher albumin secretion than control spheroids composed of Hep G2 or HuH-7 cells. Additionally, the expression levels of major CYP genes were decreased in ECM gel capsules with undiluted Matrigel (9 mg/ml) compared to those in control spheroids. However, 0.3 mg/ml Matrigel did not disrupt gene expression. Furthermore, cell polarity associated with tight junction proteins (ZO-1 and Claudin-1) and the transporter protein MRP2 was markedly induced by using 0.3 mg/ml Matrigel. Thus, high-performance three-dimensional tissues fabricated by this method are applicable to increasing the efficiency of drug screening and to regenerative medicine.

Polysaccharide Nanoparticles Bearing HDAC Inhibitor as Nontoxic Nanocarrier for Drug Delivery.

The histone deacetylase inhibitors (HDACi) are potent drugs in the treatment of inflammatory diseases and defined cancer types. However, major drawbacks of HDACi, such as valproic acid (VPA), are limited serum half-life, side effects and the short circulation time. Thus, the immobilization of VPA in a polysaccharide matrix is used to circumvent these problems and to design a suitable nanocarrier system. Therefore, VPA is covalently attached to cellulose and dextran via esterification with degree of substitution (DS) values of up to 2.20. The resulting hydrophobic polymers are shaped to spherical nanoparticles (NPs) with hydrodynamic diameter between 138 to 221 nm and polydispersity indices from 0.064 to 0.094 by nanoprecipitation and emulsification technique. Lipase treatment of the NPs leads to in vitro release of VPA and hence to an inhibition of HDAC2 activity in a HDAC2 assay. NPs are rapidly taken up by HeLa cells and mainly localize in the cytoplasm. The NPs are hemocompatible and nontoxic as revealed by the shell-less hen's egg model.