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Journal of Biotechnology Jan 2021Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream...
Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.
Topics: Diatomaceous Earth; Genetic Vectors; HEK293 Cells; Humans; Lentivirus; Transfection
PubMed: 33301854
DOI: 10.1016/j.jbiotec.2020.12.004 -
Environmental Science and Pollution... Feb 2024This research aimed to assess the effectiveness of Metarhizium robertsii, diatomaceous earth (Protect-It), and lambda-cyhalothrin, for the long-term protection of stored...
This research aimed to assess the effectiveness of Metarhizium robertsii, diatomaceous earth (Protect-It), and lambda-cyhalothrin, for the long-term protection of stored wheat against three destructive grain insect pests, Rhyzopertha dominica, Tribolium castaneum, and Trogoderma granarium. Different treatments were applied, both alone and in paired combinations in laboratory and persistence trials. Single treatments exhibited significantly lower mortality rates in comparison to the paired treatments for all tested insect species. Among the single treatments, lambda-cyhalothrin (Lamb) resulted in significantly higher mortality rates in laboratory trials, followed by diatomaceous earth (DE) and M. robertsii (Mr), with insignificant differences between Mr and DE. Evidently, DE exhibited the highest persistence after 120 days of storage for all insect species and initial exposures, although variations in mortality rates among treatments were mostly insignificant. Overall, the most effective treatment in terms of mortality in laboratory, and persistence trials, and progeny production was DE + Lamb, followed by Mr + Lamb, and Mr + DE for all tested insect species. In general, the most susceptible insect species was R. dominica, followed by T. castaneum and T. granarium. This research highlights the effectiveness of M. robertsii, DE, and lambda-cyhalothrin in providing prolonged protection of stored wheat against all the examined grain insect species.
Topics: Animals; Sheep; Insecticides; Diatomaceous Earth; Coleoptera; Insecta; Edible Grain; Nitriles; Pyrethrins
PubMed: 38294649
DOI: 10.1007/s11356-024-31824-1 -
Cells Aug 2022This paper analyses the impact of the diatomaceous earth/peat (DEP; 3:1) microbial carrier on changes in the bacterial microbiome and the development of biofilm in the...
This paper analyses the impact of the diatomaceous earth/peat (DEP; 3:1) microbial carrier on changes in the bacterial microbiome and the development of biofilm in the anaerobic digestion (AD) of confectionery waste, combined with digested sewage sludge as inoculum. The physicochemical properties of the carrier material are presented, with particular focus on its morphological and dispersion characteristics, as well as adsorption and thermal properties. In this respect, the DEP system was found to be a suitable carrier for both mesophilic and thermophilic AD. The evaluation of quantitative and qualitative changes in the genetic diversity of bacterial communities, carried out using next-generation sequencing (NGS), showed that the material has a modifying effect on the bacterial microbiome. While Actinobacteria was the most abundant cluster in the WF-control sample (WF-waste wafers), Firmicutes was the dominant cluster in the digested samples without the carrier (WF-dig.; dig.-digested) and with the carrier (WF + DEP). The same was true for the count of Proteobacteria, which decreased twofold during biodegradation in favor of Synergistetes. The cluster was identified as the most abundant genus in the two samples, particularly in WF + DEP. This information was supplemented by observations of morphological features of microorganisms carried out using fluorescence microscopy. The biodegradation process itself had a significant impact on changes in the microbiome of samples taken from anaerobic bioreactors, reducing its biodiversity. As demonstrated by the results of this innovative method, namely the BioFlux microfluidic flow system, the decrease in the number of taxa in the digested samples and the addition of DEP contributed to the microbial adhesion in the microfluidic system and the formation of a stable biofilm.
Topics: Anaerobiosis; Bacteria; Bioreactors; Diatomaceous Earth; Genetic Variation; Sewage; Soil
PubMed: 36010646
DOI: 10.3390/cells11162571 -
Materials (Basel, Switzerland) Oct 2020New composites containing a natural filler made of diatom shells (frustules), permitting the modification of polylactide matrix, were produced by Fused Deposition...
New composites containing a natural filler made of diatom shells (frustules), permitting the modification of polylactide matrix, were produced by Fused Deposition Modelling (3D printing) and were thoroughly examined. Two mesh fractions of the filler were used, one of <40 µm and the other of 40-63 µm, in order to check the effect of the filler particle size on the composite properties. The composites obtained contained diatom shells in the concentrations from 0% to 5% wt. (0-27.5% vol.) and were subjected to rheological analysis. The composites obtained as filaments of 1.75 mm in diameter were used for 3D printing. The printed samples were characterized as to hydrophilic-hydrophobic, thermal and mechanical properties. The functional parameters of the printed objects, e.g., mechanical characteristics, stability on contact with water and water contact angle, were measured. The results revealed differences in the processing behavior of the samples as well as the effect of secondary granulation of the filler on the parameters of the printing and mechanical properties of the composites.
PubMed: 33081398
DOI: 10.3390/ma13204632 -
Microorganisms Jul 2023Ticks in the USA are the most important arthropod vector of microbes that cause human and animal disease. The blacklegged tick, , the focus of this study, is able to...
Ticks in the USA are the most important arthropod vector of microbes that cause human and animal disease. The blacklegged tick, , the focus of this study, is able to transmit the bacteria that causes Lyme disease in humans in the USA. The main approach to tick control is the use of chemical acaricides and repellents, but known and potential tick resistance to these chemicals requires the discovery of new methods of control. Volcanic glass, Imergard, was recently developed to mimic the insecticide mode of action of the minerals from diatoms (diatomaceous earth, DE) for the control of malaria mosquitoes in Africa. However, studies on the use of these minerals for tick control are minimal. In a dipping assay, which was put into DE (Celite), the times of 50 and 90% death of adult female were 7.3 and 10.5 h, respectively. Our mimic of DE, Imergard, killed ticks in 6.7 and 11.2 h, respectively. In a choice-mortality assay, ticks moved onto a treated surface of Imergard and died at 11.2 and 15.8 h, respectively. Ticks had greater locomotor activity before death when treated by dipping for both Imergard and Celite versus the no-mineral control. The ticks after making contact with Imergard had the mineral covering most of their body surface shown by scanning electron microscopy with evidence of Imergard inside their respiratory system. Although the assumed mode of action of Imergard and Celite is dehydration, the minerals are not hygroscopic, there was no evidence of cuticle damage, and death occurred in as little as 2 h, suggesting minimal abrasive action of the cuticle. Semi-field and field studies are needed in the future to examine the practical use of Imergard and Celite for tick control, and studies need to examine their effect on tick breathing and respiratory retention of water.
PubMed: 37630466
DOI: 10.3390/microorganisms11081906 -
Journal of Applied Microbiology Aug 2022To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures.
AIMS
To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures.
METHODS AND RESULTS
Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates.
CONCLUSIONS
In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach.
SIGNIFICANCE AND IMPACT OF THE STUDY
Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.
Topics: Bacteria; Centrifugation; Filtration
PubMed: 35503033
DOI: 10.1111/jam.15608 -
Marine Drugs Aug 2023Diatom microalgae are a natural source of fossil biosilica shells, namely the diatomaceous earth (DE), abundantly available at low cost. High surface area, mesoporosity...
Diatom microalgae are a natural source of fossil biosilica shells, namely the diatomaceous earth (DE), abundantly available at low cost. High surface area, mesoporosity and biocompatibility, as well as the availability of a variety of approaches for surface chemical modification, make DE highly profitable as a nanostructured material for drug delivery applications. Despite this, the studies reported so far in the literature are generally limited to the development of biohybrid systems for drug delivery by oral or parenteral administration. Here we demonstrate the suitability of diatomaceous earth properly functionalized on the surface with -octyl chains as an efficient system for local drug delivery to skin tissues. Naproxen was selected as a non-steroidal anti-inflammatory model drug for experiments performed both in vitro by immersion of the drug-loaded DE in an artificial sweat solution and, for the first time, by trans-epidermal drug permeation through a 3D-organotypic tissue that better mimics the in vivo permeation mechanism of drugs in human skin tissues. Octyl chains were demonstrated to both favour the DE adhesion onto porcine skin tissues and to control the gradual release and the trans-epidermal permeation of Naproxen within 24 h of the beginning of experiments. The evidence of the viability of human epithelial cells after permeation of the drug released from diatomaceous earth, also confirmed the biocompatibility with human skin of both Naproxen and mesoporous biosilica from diatom microalgae, disclosing promising applications of these drug-delivery systems for therapies of skin diseases.
Topics: Humans; Animals; Swine; Diatoms; Microalgae; Naproxen; Diatomaceous Earth; Drug Delivery Systems; Anti-Inflammatory Agents, Non-Steroidal
PubMed: 37623719
DOI: 10.3390/md21080438 -
British Journal of Industrial Medicine Jul 1948
Topics: Diatomaceous Earth; Humans; Silicosis
PubMed: 18873553
DOI: 10.1136/oem.5.3.148 -
Marine Drugs Apr 2023Colorectal cancer is among the most prevalent and lethal cancers globally. To address this emergency, countries have developed diffuse screening programs and innovative... (Review)
Review
Colorectal cancer is among the most prevalent and lethal cancers globally. To address this emergency, countries have developed diffuse screening programs and innovative surgical techniques with a consequent decrease in mortality rates in non-metastatic patients. However, five years after diagnosis, metastatic CRC is still characterized by less than 20% survival. Most patients with metastatic CRC cannot be surgically treated. For them, the only option is treatment with conventional chemotherapies, which cause harmful side effects in normal tissues. In this context, nanomedicine can help traditional medicine overcome its limits. Diatomite nanoparticles (DNPs) are innovative nano-based drug delivery systems derived from the powder of diatom shells. Diatomite is a porous biosilica largely found in many areas of the world and approved by the Food and Drug Administration (FDA) for pharmaceutical and animal feed formulations. Diatomite nanoparticles with a size between 300 and 400 nm were shown to be biocompatible nanocarriers capable of delivering chemotherapeutic agents against specific targets while reducing off-target effects. This review discusses the treatment of colorectal cancer with conventional methods, highlighting the drawbacks of standard medicine and exploring innovative options based on the use of diatomite-based drug delivery systems. Three targeted treatments are considered: anti-angiogenetic drugs, antimetastatic drugs, and immune checkpoint inhibitors.
Topics: Animals; Nanomedicine; Diatoms; Diatomaceous Earth; Drug Delivery Systems; Nanoparticles; Colorectal Neoplasms; Antineoplastic Agents
PubMed: 37233460
DOI: 10.3390/md21050266 -
Biomaterials Advances Jul 2024Engineering of scaffolds for bone regeneration is often inspired by the native extracellular matrix mimicking its composite fibrous structure. In the present study, we...
Engineering of scaffolds for bone regeneration is often inspired by the native extracellular matrix mimicking its composite fibrous structure. In the present study, we used low loadings of diatomite earth (DE) biosilica to improve the bone regeneration potential of gelatin electrospun fibrillar microenvironments. We explored the effect of increasing the DE content from 1 % to 3 % and 5 %, respectively, on the physico-chemical properties of the fibrous scaffolds denoted FG_DE1, FG_DE3, FG_DE5, regarding the aqueous media affinity, stability under simulated physiological conditions, morphology characteristics, and local mechanical properties at the surface. The presence of biosilica generated composite structures with lower swelling degrees and higher stiffness when compared to gelatin fibers. Increasing DE content led to higher Young modulus, while the stability of the protein matrix in PBS, at 37 °C, over 21 was significantly decreased by the presence of diatomite loadings. The best preosteoblast response was obtained for FG_DE3, with enhanced mineralization during the osteogenic differentiation when compared to the control sample without diatomite. 5 % DE in FG_DE5 proved to negatively influence cells' metabolic activity and morphology. Hence, the obtained composite microfibrillar scaffolds might find application as osteoblast-responsive materials for bone tissue engineering.
Topics: Gelatin; Osteoblasts; Tissue Scaffolds; Tissue Engineering; Animals; Diatomaceous Earth; Osteogenesis; Cell Differentiation; Mice; Bone Regeneration; Cell Line; Cellular Microenvironment; Microfibrils; Extracellular Matrix
PubMed: 38796956
DOI: 10.1016/j.bioadv.2024.213894