Final Suspension Research Articles

A Comparative Study of Three Widespread Methods for Extracting Plant-Parasitic Nematodes from Soil Samples

This study was conducted in a productive farm characterized by heterogeneous soil type and planted with Balaika bananas (Musa maghrabi) in Al-Sharkia Governorate, Egypt. Three soil types namely sandy, sandy clay, and clayey soil were determined in this farm. Three methods were selected for extracting plant-parasitic nematodes present in the previous soil types. These methods were extraction trays (ET), decanting plus sieving (DS) and centrifugal flotation (CF) methods. Results showed that the centrifugal flotation was the most efficient extraction method. The relative efficacy of the extraction (%) in the DS method was 36.76 and 39.29% in sandy clay and clayey soil, respectively while, in the CF method it was 35.94% in clayey soil type, for genus Meloidogyne. The inactive nematode genera e.g. Criconemoides was not extracted by ET and DS methods in sandy and sandy clay soil types. The most efficient method for extracting this genus was the CF method it recorded 51.82 and 47.77% in sandy and clayey soil types, respectively. In this comparative study it could be concluded that the centrifugal flotation method (CF) was the most efficient method for extraction the sluggish and motile nematodes, take short time for extraction, and the final suspension was clear. On the other hand, the ET method using a small amount of water had an importance today in water management strategy.

Development and evaluation of an omeprazole-based delayed-release liquid oral dosage form.

Modified-release oral dosage forms are commonly used in pharmaceutics to delay or sustain the release of drugs. Nowadays, they are only marketed as solid dosage forms such as capsules or tablets. Therefore, the development of a liquid oral dosage form with modified-release properties has been keenly awaited to increase the compliance of patients with a swallowing impairment, such as paediatric, older or critically ill patients. In this study, a new technology has been developed that consists of multi-layered particles suspended extemporaneously in a syrup, using omeprazole as a model drug. The coating procedure was optimized to obtain a yield of minimum 90% w/w and a mean diameter below 500 µm. Eudragit® E100 and Eudragit® L100-55 were used to prevent the early release of omeprazole in the syrup and in the acidic environment of the stomach, respectively. These polymers allowed the stability of the coated particles to be ensured when dispersed in a liquid and the enteric release of the drug to be targeted. It was demonstrated that our new system presented similar release performances to existing marketed enteric dosage forms. It is able to protect omeprazole for 2 h in acidic medium at pH 1.2, while omeprazole was entirely released at pH 6.8 within 45 min. Once the final suspension is prepared extemporaneously, it presents sufficient stability to guarantee the administration of multiple doses filled into a syrup bottle and kept for a limited storage time at room temperature (e.g. up to 10 doses to be administered within 10 days).

Modification of Ceria Nanoparticles by Accelerated Electron Irradiation

The luminescence and optical properties of ceria (CeO2) nanoparticles irradiated by accelerated electrons with an energy of 10 MeV are investigated in this work. CeO2 nanoparticles are prepared by the pulse electron evaporation of a ceramic target with vapor condensation of the evaporated material in a low-pressure gas on a NANOBIM-2 installation. The irradiation of CeO2 nanopowders is found to cause the degradation of their luminescence and optical characteristics. The irradiation of a final aqueous suspension leads to the shift of a photoluminescence band toward the long wave range spectral range in comparison with the irradiation of a nanopowder. The catalytic-like behavior of CeO2 nanoparticles interacting with hydrogen peroxide is analyzed, as well. Nanoparticles modified by the electron beam exhibit the deterioration of their enzyme-like activity.

Erratum: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification.

Erratum: Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification.

Control of liposomes diameter at micrometric and nanometric level using a supercritical assisted technique

Supercritical assisted Liposome formation process (SuperLip) allows one-shot, continuous and reproducible production of liposomes. In this work, the optimization of Gas to Liquid Ratio of the Expanded Liquid (GLR-EL); i.e., the ratio between carbon dioxide flow rate and ethanol flow rate, is presented. It allows to control liposomes diameter from micrometric to nanometric range and the amount of Solvent Residue (SR) in the final suspension. Working at a GLR-EL lower than about 1.8, resulted in the production of micrometric and sub-micrometric L-α-phosphatidylcholine (PC) liposomes, with mean diameters ranging from 1729 ± 733 nm to 878 ± 170 nm; whereas, working at larger GLRs-EL nanometric liposomes, with diameters down to about 139 ± 49 nm were produced. Liposomes produced at GLR-EL 6.0 were loaded with a hydrophilic compound: an antibiotic, vancomycin, or a lipophilic antioxidant, farnesol. Liposome diameters were again nanometric down to 126 ± 35 nm for farnesol loaded vesicles and encapsulation efficiencies (EE) were up to 76.7 ± 1.9%. The mean size of vancomycin loaded liposomes was 250 ± 93 nm and EE up to 74.0 ± 1.1% were obtained. SR down to 10 ppm were obtained on loaded vesicles, improving the possibility of their use for pharmaceutical applications.

A focus of the nanoprecipitation by solvent displacement: example of poly(MAOTIB) intended to in vivo applications

Through this study, we propose to specifically focus on a particular stage of the fabrication of polymeric nanoparticles intended to be used as contrast agent for biomedical X-ray imaging. These nanoparticles, made from nanoprecipitation of preformed polymer, poly(MAOTIB) (poly(2-methacryloyloxyethyl(2,3,5-triiodobenzoate))) follow a solvent displacement process. This method, widely used in literature, is sensitive to the formulation and process parameters such as nature and concentrations of surfactant and polymer, solvent / non-solvent ratio, rate of addition of one phase in the other one, respective volumes of the phase, and homogenization shearing rate. On the other hand, in function of the aimed administration route, the final suspension should obey to specific constraints on final product, e.g. size range and polydispersity, final particle concentration (i.e. iodine concentration) and surfactant concentration. In the present work, we report a specific investigation on the nanoprecipiation of poly(MAOTIB) in tetrahydrofuran, dropped in water or ethanol (as non-solvent) and stabilized by nonionic surfactant. The objective is to show and explain the potentials and limitations of such the process, but also to provide a guidance on the way to optimize it.

Safety and efficacy evaluations of an adeno-associated virus variant for preparing IL10-secreting human neural stem cell-based therapeutics.

Gene therapy technologies are inevitably required to boost the therapeutic performance of cell therapies; thus, validating the efficacy of gene carriers specifically used for preparing cellular therapeutics is a prerequisite for evaluating the therapeutic capabilities of gene and cell combinatorial therapies. Herein, the efficacy of a recombinant adeno-associated virus derivative (rAAVr3.45) was examined to evaluate its potential as a gene carrier for genetically manipulating interleukin-10 (IL10)-secreting human neural stem cells (hNSCs) that can potentially treat ischemic injuries or neurological disorders. Safety issues that could arise during the virus preparation or viral infection were investigated; no replication-competent AAVs were detected in the final cell suspensions, transgene expression was mostly transient, and no severe interference on endogenous gene expression by viral infection occurred. IL10 secretion from hNSCs infected by rAAVr3.45 encoding IL10 did not alter the transcriptional profile of any gene by more than threefold, but the exogenously boosted IL10 was sufficient to provoke immunomodulatory effects in an ischemic brain injury animal model, thereby accelerating the recovery of neurological deficits and the reduction of brain infarction volume. This study presents evidence that rAAVr3.45 can be potentially used as a gene carrier to prepare stem cell therapeutics.

Модификация наночастиц оксида церия при облучении ускоренными электронами

AbstractThe luminescence and optical properties of ceria (CeO_2) nanoparticles irradiated by accelerated electrons with an energy of 10 MeV are investigated in this work. CeO_2 nanoparticles are prepared by the pulse electron evaporation of a ceramic target with vapor condensation of the evaporated material in a low-pressure gas on a NANOBIM-2 installation. The irradiation of CeO_2 nanopowders is found to cause the degradation of their luminescence and optical characteristics. The irradiation of a final aqueous suspension leads to the shift of a photoluminescence band toward the long wave range spectral range in comparison with the irradiation of a nanopowder. The catalytic-like behavior of CeO_2 nanoparticles interacting with hydrogen peroxide is analyzed, as well. Nanoparticles modified by the electron beam exhibit the deterioration of their enzyme-like activity.

Inertial-Based Filtration Method for Removal of Microcarriers from Mesenchymal Stem Cell Suspensions

Rapidly evolving cell-based therapies towards clinical trials demand alternative approaches for efficient expansion of adherent cell types such as human mesenchymal stem cells (hMSCs). Using microcarriers (100–300 µm) in a stirred tank bioreactor offers considerably enhanced surface to volume ratio of culture environment. However, downstream purification of the harvested cell product needs to be addressed carefully due to distinctive features and fragility of these cell products. This work demonstrates a novel alternative approach which utilizes inertial focusing to separate microcarriers (MCs) from the final cell suspension. First, we systematically investigated MC focusing dynamics inside scaled-up curved channels with trapezoidal and rectangular cross-sections. A trapezoidal spiral channel with ultra-low-slope (Tan(α) = 0.0375) was found to contribute to strong MC focusing (~300 < Re < ~400) while managing high MC volume fractions up to ~1.68%. Accordingly, the high-throughput trapezoidal spiral channel successfully separated MCs from hMSC suspension with total cell yield~94% (after two passes) at a high volumetric flow rate of ~30 mL/min (Re~326.5).

Development of a transportable single-use biomanufacturing system for therapeutic phage preparations

Background: The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. Product discovery costs are very low, but conventional biopharmaceutical production of phages is lengthy, complex and costly. In addition, phage treatment in low-income countries with poor infrastructure, battlefields, healthcare emergency situations (e.g. the 2012 foodborne E. coli 0104:H4 outbreak in Germany) demands a fast, global and decentralized access to phages. To address this demand, we developed a single-use, pre-sterilised, portable and closed biomanufacturing platform for therapeutic phage preparations. Methods & Materials: The basic components of this platform are as follows: a stacked bioreactor vessel with an adaptable number of chambers, in which the host bacterial strain, phage seed, culture medium (soft agar overlay technique) and extraction buffer are injected, is connected to (a) a cascade of clarification, purification and sterilisation filters, (b) phage vent filters, (c) tubings and connectors and (d) a filling system to dispense the final phage suspension into recipients. The following devices are necessary: a peristaltic pump, a fridge, a shaker and an incubator. Twenty-four different prototype assemblies for the production of Pseudomonas aeruginosa phages were tested. The following parameters were measured: pressure in function of volumetric throughput, phage yield and titre, endotoxin level, pH and assembly integrity (leaks). Results: The most suitable of the 24 tested prototype assemblies (presented at the conference) allowed for the production of 1-200 L (per bioreactor chamber) of phage suspension at a concentration of 107 pfu/ml with endotoxin levels and pH compatible with intravenous (IV) application, and this in 24 to 36 h. Conclusion: We developed a small-scale, portable and disposable biomanufacturing platform that allows for bacterial infection treatment at point-of-care, in short time frames, and at low cost, especially useful in remote locations with limited infrastructure since it does not necessarily require a cleanroom environment. MM and HVR contributed equally to this work. Funding: Grant HFM 15-5 of the Royal Higher Institute for Defense.

Depletion of Dead Cells from Primary Tissue in 6 Minutes

Abstract Tissue specific research often requires mechanical and/or enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process resulting in a significant number of dead cells in the final cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of primary tissues. During apoptosis, the cell membrane loses its phospholipid asymmetry resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. By targeting exposed PS with Annexin V we have developed a rapid method (EasySep™) to immunomagnetically remove dead cells from primary tissue samples. Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from an initial viability of 39.6 ± 12.3% AnxV−/PI− to 70.9 ± 11.8% AnxV−/PI. From 1×10^8 total start cells, 1.43 ± 0.68 ×10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes cultured overnight, viability was improved from 23.7 ± 9.8% AnxV−/PI− to 67.7 ± 12.1% AnxV−/PI− with recovery of 1.27 ± 0.52 ×10^7 live cells from 1×10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.

α‐Alumina nanospheres from nano‐dispersed boehmite synthesized by a wet chemical route

AbstractIn this work, stable aqueous suspensions of nano‐boehmite were developed through a hybrid wet‐chemical route that uses hyperbranched dendritic poly(ethylene)imine (PEI) as template material for boehmite formation aiming at the development of a deagglomerated α‐alumina nanopowder after calcination. The method involves firstly the interaction between the Al precursor and PEI followed by the hydrolysis and polycondensation reactions. The study was aiming to investigate the effect of solids content and pH during reactions on both the stability of the final suspension and the morphology of the resulting nanocrystals. For this purpose, the suspensions were evaluated through viscosity measurements, zeta potential analyses, FT‐IR, DLS and sedimentation studies, whereas after the proper centrifugation, drying, and calcination steps, the as‐received nanocrystals were evaluated through SEM, TEM and XRD studies. In addition, the boehmite nanopowder was studied using Thermogravimetric and Differential Thermal Analysis, whereas its sinterability was evaluated by dilatometric measurements. The investigation showed that the conditions employed affect greatly both the morphology of nanocrystals as well as the dispersion and the stability of the suspensions. The boehmite suspension with the optimum dispersion and stability can lead, after calcination at 1050°C, to a fine deagglomerated α‐alumina nanopowder with a mean size at about 10 nm.

The Critical Role of Thioacetamide Concentration in the Formation of ZnO/ZnS Heterostructures by Sol-Gel Process.

ZnO/ZnS heterostructures have emerged as an attractive approach for tailoring the properties of particles comprising these semiconductors. They can be synthesized using low temperature sol-gel routes. The present work yields insight into the mechanisms involved in the formation of ZnO/ZnS nanostructures. ZnO colloidal suspensions, prepared by hydrolysis and condensation of a Zn acetate precursor solution, were allowed to react with an ethanolic thioacetamide solution (TAA) as sulfur source. The reactions were monitored in situ by Small Angle X-ray Scattering (SAXS) and UV-vis spectroscopy, and the final colloidal suspensions were characterized by High Resolution Transmission Electron Microscopy (HRTEM). The powders extracted at the end of the reactions were analyzed by X-ray Absorption spectroscopy (XAS) and X-ray diffraction (XRD). Depending on TAA concentration, different nanostructures were revealed. ZnO and ZnS phases were mainly obtained at low and high TAA concentrations, respectively. At intermediate TAA concentrations, we evidenced the formation of ZnO/ZnS heterostructures. ZnS formation could take place via direct crystal growth involving Zn ions remaining in solution and S ions provided by TAA and/or chemical conversion of ZnO to ZnS. The combination of all the characterization techniques was crucial to elucidate the reaction steps and the nature of the final products.

A protocol to isolate and qualify purified human preantral follicles in cases of acute leukemia, for future clinical applications

BackgroundAutotransplantation of cryopreserved ovarian cortex can be associated with a risk of cancer cell reseeding. This issue could be eliminated by grafting isolated preantral follicles. Collagenase NB6 is an enzyme produced under good manufacturing practices (GMP) in compliance with requirements for tissue engineering and transplantation in humans and thus can be used to isolate preantral follicles from ovarian tissue in the framework of further clinical applications. Multicolor flow cytometry is an effective tool to evaluate the potential contamination of follicular suspensions by leukemic cells.MethodsThe efficiency of collagenase NB6 was evaluated in comparison to collagenase type IA and Liberase DH, in terms of yield, morphology and viability. A short-term in vitro culture of follicles isolated with collagenase NB6 was conducted for 3 days in a fibrin matrix. A modelization procedure was carried out to detect the presence of leukemic cells in follicular suspensions using multicolor flow cytometry (MFC).ResultsNo statistical differences were found between collagenase NB6, Liberase DH (p = 0.386) and collagenase type IA (p = 0.171) regarding the number of human preantral follicles isolated. The mean diameter of isolated follicles was significantly lower with collagenase NB6 (p < 0.0001). The survival rate of isolated follicles was 93.4% (n = 272) using collagenase NB6 versus 94.9% (n = 198) with Liberase DH and 92.6% (n = 298) using collagenase type IA. Even after 3 days of in vitro culture in a fibrin scaffold, most of the isolated follicles were still alive after using collagenase NB6 (90.7% of viable follicles; n = 339). The rate of isolated Ki67-positive follicles was 29 ± 9.19% before culture and 45 ± 1.41% after 3 days. In 23 out of 24 follicular suspensions analyzed, the detection of leukemic cells by MFC was negative. The purification had no significant impact on follicle viability.ConclusionThe isolation and purification of human preantral follicles were performed following good manufacturing practices for cell therapy. Multicolor flow cytometry was able to confirm that final follicular suspensions were free from leukemic cells. This safe isolation technique using collagenase NB6 can be considered for future clinical applications.

SELECTED EPIDEMICS & EMERGING PATHOGENS – PREVENTIVE STRATEGIES

The aim of this nonrandomized, open label, phase 1 clinical trial was to evaluate the safety and the feasibility of the treatment with autologous bone marrow–derived endothelial progenitor cells (EPC) in decompensated liver cirrhosis. In addition, the changes in liver function and hepatic venous pressure gradient (HVPG) and their relation with the characteristics of the cellular product were analyzed. Twelve patients with Child-Pugh ≥8 liver cirrhosis underwent bone marrow harvest for ex vivo differentiation of EPC. The final product was administered through the hepatic artery in a single administration. Patients underwent clinical and radiologic follow-up for 12 months. The phenotype and the ability to produce cytokines and growth factors of the final cellular suspension were analyzed. Eleven patients were treated (feasibility 91%). No treatment-related severe adverse events were observed as consequence of any study procedure or treatment. Model for end-stage liver disease score improved significantly (P 0.042) in the first 90 days after cells administration and 5 of the 9 patients alive at 90 days showed a decreased of HVPG. There was a direct correlation between the expression of acetylated-low density lipoprotein and von Willebrand factor in the cellular product and the improvement in liver function and HVPG. The treatment with EPCs in patients with decompensated liver cirrhosis is safe and feasible and might have therapeutic potential. Patients receiving a higher amount of functionally active EPC showed an improvement of liver function and portal hypertension suggesting that the potential usefulness of these cells for the treatment of liver cirrhosis deserves further evaluation.

Nanofat 2.0: experimental evidence for a fat grafting rich in mesenchymal stem cells.

Different strategies have been developed in the last decade to obtain fat grafts as rich as possible of mesenchymal stem cells, so exploiting their regenerative potential. Recently, a new kind of fat grafting, called "nanofat", has been obtained after several steps of fat emulsification and filtration. The final liquid suspension, virtually devoid of mature adipocytes, would improve tissue repair because of the presence of adipose mesenchymal stem cells (ASCs). However, since it is probable that many ASCs may be lost in the numerous phases of this procedure, we describe here a novel version of fat grafting, which we call "nanofat 2.0", likely richer in ASCs, obtained avoiding the final phases of the nanofat protocol. The viability, the density and proliferation rate of ASCs in nanofat 2.0 sample were compared with samples of nanofat and simple lipoaspirate. Although the density of ASCs was initially higher in lipoaspirate sample, the higher proliferation rate of cells in nanofat 2.0 virtually filled the gap within 8 days. By contrast, the density of ASCs in nanofat sample was the poorest at any time. Results show that nanofat 2.0 emulsion is considerably rich in stem cells, featuring a marked proliferation capability.

Al2O3\u2013TiO2/ZrO2\u2013SiO2 based porous ceramics from particle-stabilized wet foam

The porous ceramics based on Al2O3–TiO2/ZrO2–SiO2 from particle-stabilized wet foam by direct foaming were discussed. The initial Al2O3–TiO2 suspension was prepared by adding TiO2 suspension to partially hydrophobized colloidal Al2O3 suspension with equimolar amount, to form Al2TiO5 on sintering. The secondary ZrO2–SiO2 suspension was prepared using the equimolar composition, and to obtain ZrSiO4, ZrTiO4, and mullite phases in the sintered samples, the secondary suspension was blended into the initial suspension at 0, 10, 20, 30, and 50 vol%. The wet foam exhibited an air content up to 87%, Laplace pressure from 1.38 to 2.23 mPa, and higher adsorption free energy at the interface of approximately 5.8×108 to 7.5×108 J resulting an outstanding foam stability of 87%. The final suspension was foamed, and the wet foam was sintered from 1400 to 1600 °C for 1 h. The porous ceramics with pore size from 150 to 400 μm on average were obtained. The phase identification was accomplished using X-ray diffraction (XRD), differential thermal analysis (DTA), and thermogravimetric analysis (TGA), and microstructural analysis was performed using field emission scanning electron microscopy (FESEM).

Synthesis of nanocomposite coating based on TiO&lt;sub&gt;2&lt;/sub&gt;/ZnAl layer double hydroxides

The aim of this investigation was the synthesis of nanocomposite coatings based on Zn-Al layered double hydroxides (Zn-Al LDH) and TiO2. The Zn-Al LDH material, which acted as the catalyst support of the active TiO2 component (in the content of 3 and 10 wt. %), was synthesized by a low super saturation co-precipitation method. The interaction between the Zn-Al LDH and the active TiO2 component was accomplished by using vacuum evaporation prior to the mechanical activation and only by mechanical activation. The final suspension based on Zn-Al LDH and 10wt. % TiO2, impregnated only by mechanical activation, showed the optimal characteristics from the aspect of particle size distribution and XRD analysis. These properties had a positive effect on the functional properties of the coatings (photocatalytic activity and self-cleaning efficiency) after the water rinsing procedure.

Are universities ready for interculturality? The case of the Intercultural University ‘Amawtay Wasi’ (Ecuador)

This article focuses on the creation of the Intercultural University ‘Amawtay Wasi’ in 2003, and its evolution up until its final suspension from the university system in Ecuador. I reflect on the difficulty of implementing educational approaches based on locally situated cultural identities that involve epistemologies different from those which are hegemonic in academic spheres on the global level. From the point of view of the indigenous movement, the current situation is solely the result of the ignorance and arrogance of the state, which ‘has led to a backlash against the gains made by indigenous people that has even led to a decline in terms of indigenous rights’, calling the current government a government of ‘a new colonization’ (Salvador). However, interviews conducted in the years after the closure of Amawtay Wasi bring to light certain discourses and opposition that go beyond the obvious intellectual and political reluctance of the current government to maintain the Amawtay Wasi Intercultural University.

Assembly of nanoparticles-polyelectrolyte complexes in nanofiber cellulose structures

We report the effect of cationic polyacrylamide (CPAM) addition on the structural assembly of silica nanoparticles (NPs) within a nanocellulose fibre matrix. Paper like composites are fabricated by first forming complexes of NPs with CPAM, then adding those to a suspension of nanocellulose fibres; followed by filtration, pressing and drying of the final suspension. Complementary small angle X-ray scattering (SAXS) and microscopy (SEM, TEM) investigations of these composites showed a lognormal bimodal distribution of NP sizes. Data analysis allows understanding interparticle interactions within assemblies of SiO2 NPs at the nanometer scale with respect to different dosage of CPAM. Increasing CPAM dosage increases retention of NPs within the cellulose matrix with stronger interparticle interactions and produces composites with smaller pores. The correlation length of NPs, indicative of the size of the NP clusters increased from 30 to 70nm as the CPAM dosage increased from 16.5 to 330mg/g NPs. Retention and assembly of SiO2 NPs by varying CPAM dosage results from the balance of different interaction forces between NPs, CPAM and nanocellulose fibres. Understanding the effect of CPAM dosage on the various NP and composite structural conformations enables us to engineer novel hierarchically and functional cellulose based structured materials.