Comparative study of three novel ion exchange resins with DEAE-cellulose for the purification of Trypanosoma evansi.

Ion exchange chromatography is a method that uses the different surface charges of trypanosomes and blood cells to separate them. This makes it possible to use molecular and immunological methods to diagnose or study these protozoans. DEAE-cellulose resin is commonly used to perform this method. The goal of this study was to compare three novel chromatographic resins designated as PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®). The resins were evaluated based on their ability to isolate the parasite, purification time, examination of parasite viability and morphology, and trypanosome recovery potential after passing through the columns. In terms of the evaluated parameters, there was no significant difference between DEAE-cellulose and the three tested resins in most experiments. However, PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®) resins are less expensive and easier to prepare than DEAE-Cellulose, making them an alternative for the purification of Trypanosoma evansi.

Biofabrication of skin tissue constructs using alginate, gelatin and diethylaminoethyl cellulose bioink.

INTRODUCTION: Biofabrication of skin tissue equivalents using 3D bioprinting technology has gained much attention in recent times due to the simplicity, the versatility of the technology and its ability in bioengineering biomimetic tissue histology. The key component being the bioink, several groups are actively working on the development of various bioink formulations for optimal skin tissue construction. METHODS: Here, we present alginate (ALG), gelatin (GEL) and diethylaminoethyl cellulose (DCEL) based bioink formulation and its application in bioprinting and biofabrication of skin tissue equivalents. Briefly, DEAE cellulose powder was dispersed in alginate solution with constant stirring at 60 °C to obtain a uniform distribution of cellulose fibers; this was then mixed with GEL solution to prepare the bioink. The formulation was systematically characterized for its morphological, physical, chemical, rheological, biodegradation and biocompatibility properties. The printability, shape fidelity and cell-laden printing were assessed using the CellInk bioprinter. RESULTS: The bioink proved to be a good printable, non-cytotoxic and stable hydrogel formulation. The primary human fibroblast and keratinocyte-loaded 3D bioprinted constructs showed excellent cell viability, collagen synthesis, skin-specific marker and biomimetic tissue histology. CONCLUSION: The results demonstrated the successful formulation of ALG-GEL-DCEL bioink and its application in the development of human skin tissue equivalents with distinct epidermal-dermal histological features.

Stabilization of Glycosylated ß-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

Structural characterization and induced copper stress resistance in rice of exopolysaccharides from Lactobacillus plantarum LPC-1.

Excessive accumulation of copper could decrease growth and quality of crops, and little information was currently available on the role of exopolysaccharides (EPS) from Lactobacillus plantarum in inducing copper stress resistance in plants. The main objective of this work was to purify and characterize the EPS produced by our isolated L. plantarum LPC-1, and evaluate its potential protection for rice against copper stress. Firstly, two fractions (EPS-1 and EPS-2) were separated and purified from L. plantarum LPC-1 by DEAE-52 cellulose and Sephadex G-100 cellulose column chromatography. According to the further scanning electron microscope (SEM), ultraviolet-visible (UV), fourier-transform infrared spectroscopy (FTIR) spectroscopy and gas chromatography (GC) analyses, it was observed that EPS-1 and EPS-2 were heteropolysaccharides that were composed of mannose and glucose with molar ratio of 2.40:15.01 and 3.02:11.63, respectively. Additionally, the two fractions possessed considerable antioxidant activities, and EPS-1 had a stronger antioxidant activity than EPS-2 in vitro. Furthermore, exogenous addition of EPS significantly alleviated the toxic effects by copper on rice seedlings. In conclusion, this study provided evidence of the EPS-mediated reduction of copper toxicity in rice seedlings at physiological and biochemical levels, suggesting that EPS could be considered as novel and effective plant immune inducers in crops.

Structure analysis of polysaccharides purified from Cyclocarya paliurus with DEAE-Cellulose and its antioxidant activity in RAW264.7 cells.

CPP was isolated from Cyclocarya paliurus (C. paliurus) and CPP-D was purified from CPP with a further step by DEAE-Cellulose. In this study, the structure and antioxidant activities of these two polysaccharides were investigated. The molecular weight of CPP was determined as 1.15 × 105 Da and the monosaccharides of it were Rha, Ara, Xyl, Man, Glc, Gal in a molar ratio of 0.021:0.237:0.020:0.036:0.454:0.231, while the molecular weight of CPP-D was 9.1 × 103 Da and the monosaccharides of it were Man, Glc, Gal in a molar ratio of 0.235:0.677:0.088. CPP-D consisted of three structural residues →4)-ß-D-Glc-(1→, →2,6)-ß-D-Man-(1→ and →4)-ß-D-Gal. These structures were characterized by SEM, FT-IR, GC-MS, HPGPC, and NMR. The antioxidant assay in RAW264.7 cell showed that both CPP and CPP-D promoted cell viability and antioxidant activity, which decreased the content of MDA and increased the activity of SOD, T-AOC, CAT (P < .05). As a result, CPP-D isolated by DEAE-Cellulose didn't reduce the antioxidant activity of C. paliurus polysaccharide and could enhance the cell viability.

Chemical structure and inhibition on α-glucosidase of polysaccharide with alkaline-extracted from glycyrrhiza inflata residue.

A new neutral polysaccharide, named AGP, was extracted from glycyrrhiza residue by 5% NaOH alkaline solution and purified by DEAE-celluloseand Sephadex G-150. A single and symmetrical peak was shown by HPLC, indicating that AGP is a homogeneous polysaccharide with a molecular weight of 2.89 × 103 KDa. Thespecific rotation of AGP was detected by a polarimeter and it was +45°. Monosaccharide composition analysis indicated that AGP was consisted of l-rhamnose: l-arabinose: d-xylose: d-mannose: d-glucose and d-galactose with a molar ratio of 1:2.33:2.85:0.69:3.05:1.54. The structure of AGP was analyzed by GC-MS, periodate oxidation, Smith degradation, FT-IR, methylation and NMR, which indicated that the AGP was composed of → 6)-ß-d-Glcp-( â†’ backbone and the â†’4)-α-d-Xylp-(1→, →5)-α-l-Araf-(1→, →3)-α-l-Rhap-(1→, →6)-α-d-Galp-(1→, →3,6)-α-Manp-(1→ and →1)-ß-d-Glcp as branches. The results of Congo red experiment and circular dichroism (CD) showed that there was triple helix conformation in AGP. The micro-structure of AGP were detected by scanning electron microscopy (SEM), which concluded that the shape of AGP was a "thin slice" and its structure is not regular. The crystal configuration was identified by X-ray diffraction (XRD), showing that there is no crystal structure. Furthermore, the AGP exhibited certain inhibition activity on α-glucosidase.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns).

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations. HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity. Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal. These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Structure elucidation and bioactivities of a new polysaccharide from Xiaojin Boletus speciosus Frost.

A new water-soluble polysaccharide named BSF-X was extracted and purified from the fruiting bodies of Boletus speciosus Frost which had a molecular weight of 141,309 Da. The structure identification results showed that BSF-X was mainly composed of ß-d-glucose and α-D-galactose. BSF-X had a backbone of 1, 4-linked ß-d-glucose of which branches were mainly composed of two 1, 6-linked α-D-galactose residue and a 4-linked ß-d-glucose at the end of the branches. Antitumor activities results showed that BSF-X could inhibit the proliferation of L929 cells in vitro and S180 tumor cells in vivo. Immunoregulatory activities results showed that BSF-X could promote the proliferation of T cells, B cells and macrophages by promoting the cells into S phase from G0/G1 phase. Polysaccharide BSF-X can also enhance the phagocytes is and cytokine secretion of macrophages. This study introduced the new polysaccharide BSF-X as a valuable source which exhibit unique antitumor and immunoregulatory properties.

Physicochemical properties and cell-based bioactivity of Pu'erh tea polysaccharide conjugates.

Polysaccharide conjugates were prepared from Pu'erh tea and fractionated by DEAE-cellulose DE-52 column chromatography to yield one unexplored polysaccharide-conjugate fraction termed TPC-P with a molecular weight of 251,200Da. DVS (dynamic vapour sorption) result discovered that the humidity condition of long-term preservation for TPC-P is below 70% RH. Although it contained proteins, TPC-P could not bind to the Coomassie Brilliant Blue dyes G250 and R250. The "shoulder-shaped" ultroviolet absorption peak in TPC-P UV-vis scanning spectum ascribe theabrownins that inevitably adsorbed the polysaccharide conjugate. Zeta potential results demonstrated TPC-P aqueous solution merely presented the negative charge properties of polysaccharides instead of acid-base property of its protein section, and had more stability in greater than pH 5.5. No precipitation or haze occurred in the three TPC-P/EGCG aqueous mixtures during their being stored for 12h. The phase separation was observed in aqueous mixtures of TPC-P and type B gelatin. TPC-P possessed the fine stability as a function of temperature heating and cooling between 0 and 55°C. It is proposed that some properties of the covalent binding protein of TPC-P were "shielded" by its polysaccharide chains.

Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 µM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 µM and 2.5 µM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 µM.

Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

Nanofiber adsorbents for high productivity continuous downstream processing.

An ever increasing focus is being placed on the manufacturing costs of biotherapeutics. The drive towards continuous processing offers one opportunity to address these costs through the advantages it offers. Continuous operation presents opportunities for real-time process monitoring and automated control with potential benefits including predictable product specification, reduced labour costs, and integration with other continuous processes. Specifically to chromatographic operations continuous processing presents an opportunity to use expensive media more efficiently while reducing their size and therefore cost. Here for the first time we show how a new adsorbent material (cellulosic nanofibers) having advantageous convective mass transfer properties can be combined with a high frequency simulated moving bed (SMB) design to provide superior productivity in a simple bioseparation. Electrospun polymeric nanofiber adsorbents offer an alternative ligand support surface for bioseparations. Their non-woven fiber structure with diameters in the sub-micron range creates a remarkably high surface area material that allows for rapid convective flow operations. A proof of concept study demonstrated the performance of an anion exchange nanofiber adsorbent based on criteria including flow and mass transfer properties, binding capacity, reproducibility and life-cycle performance. Binding capacities of the DEAE adsorbents were demonstrated to be 10mg/mL, this is indeed only a fraction of what is achievable from porous bead resins but in combination with a very high flowrate, the productivity of the nanofiber system is shown to be significant. Suitable packing into a flow distribution device has allowed for reproducible bind-elute operations at flowrates of 2,400 cm/h, many times greater than those used in typical beaded systems. These characteristics make them ideal candidates for operation in continuous chromatography systems. A SMB system was developed and optimised to demonstrate the productivity of nanofiber adsorbents through rapid bind-elute cycle times of 7s which resulted in a 15-fold increase in productivity compared with packed bed resins. Reproducible performance of BSA purification was demonstrated using a 2-component protein solution of BSA and cytochrome c. The SMB system exploits the advantageous convective mass transfer properties of nanofiber adsorbents to provide productivities much greater than those achievable with conventional chromatography media.

Extraction, characterization and antimicrobial activity of sulfated polysaccharides from fish skins.

Sulfated polysaccharides were extracted from gray triggerfish (GTSP) and smooth hound (SHSP) skins. Their chemical and physical characteristics were determined using X-ray diffraction and Infrared spectroscopic analysis. The antibacterial activities of GTSP and SHSP against Listeria monocytogenes (ATCC 43251), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 43972) and Enterobacter sp were evaluated by determining clear growth inhibition zone diameters and the minimum inhibitory concentration (MIC) values and by essays in liquid media. GTSP and SHSP were fractionated by a Diethylaminoethyl-cellulose chromatography. Fraction FGII, from GTSP, and fraction FSII, from SHSP, showed the most important inhibitory effects against the tested bacterial species. The sulfated polysaccharides from fish skins did not show hemolytic activity towards bovine erythrocytes. Overall, the results suggested that those polysaccharides could offer promising sources of polysaccharides for future application as dietary ingredients in the nutraceutical industry.

3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].

BACKGROUND: Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS: hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. RESULTS: hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. CONCLUSION: This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions.

Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein-metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel 'tentacle' carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0-8.0 and temperature optimum at 25°C. Immobilized PPO shows almost 100% of activity at 0°C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8h duration.

Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification.

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Cryopreservation of Trypanosoma evansi after DEAE-cellulose purification: Evaluation of infective parameters.

Cryopreservation is a method of keeping parasites alive in a laboratory. However, this technique may also damage the parasite. Alternatively, parasites may be maintained by in vitro culture. Unfortunately, for Trypanosoma evansi no effective medium that is able to maintain the parasite for more than 4 months has been described. In this study, we examined the effect of purifying trypomastigote through DEAE-cellulose chromatography before and after cryopreservation, by analyzing the pre-patent period, longevity, parasitemia, and count of viable parasites. Our results showed a three-times increase in the concentration of viable trypomastigote in DEAE-purified cryopreserved parasites as compared to non-DEAE-purified cryopreserved parasites. This indicates that DEAE-cellulose chromatography followed by cryopreservation is an effective method for the storage and preservation of T. evansi, with the advantage that the stocked parasites will be ready to use in molecular biology procedures.

Interaction of cellulose-based cationic polyelectrolytes with mucin.

Mucoadhesivity of water-soluble polymers is an important factor, when testing their suitability for controlled drug delivery systems. For this purpose, the interaction of new cationic cellulose polyelectrolytes with lyophilized mucin was investigated by means of turbidimetric titration, microscopy and measurement of zeta potential and particle size changes in the system. Results show that the cellulose derivatives interact with mucin. This interaction became stronger if cellulose macromolecules contained positively charged groups and an electrostatic interaction with the negatively charged mucin particles occurred. Under certain conditions flocculation of mucin particles by the cellulose polyelectrolyte was observed.

Immobilization of phenylalanine dehydrogenase and its application in flow-injection analysis system for determination of plasma phenylalanine.

Phenylalanine dehydrogenase (L-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized L-PheDH was shifted from pH 10.4 to 11.0. The immobilized L-PheDH showed activity variations close to the maximum value in a wider temperature range of 45-55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized L-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K (m) values of the native and the immobilized L-PheDH were determined as K(m Phe) = 0.118, 0.063 mM and K(m NAD)(+) = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5-600 µM Phe at 9 mM NAD(+) with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.

Decontamination of polymerase chain reaction reagents using DEAE-cellulose.

Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity. We also show the suitability of the proposed approach for decontamination of the Taq polymerase, monoclonal antibodies to Taq polymerase, and Moloney murine leukemia virus (M-MLV) reverse transcriptase.