Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.

Viral Filtration: A Review of Current and Future Practices in Bioprocessing

For drug products manufactured in mammalian cells, safety assurance practices are needed during production to assure that the final medicinal product is safe from the potential risk of viral contamination. Virus filters provide viral retention for a range of viruses through robust, size-based retention mechanism. Therefore, a viral filtration step is commonly utilized in a well-designed recombinant therapeutic protein purification process and is a key component in an overall strategy to minimize the risks of adventitious and endogenous viral particles during the manufacturing of biotechnology products. This review summarizes the history of viral filtration, currently available viral filters and prefilters, and viral filtration integrity test methods and study models. There is also discussion of current understanding and gaps with an eye toward future trends and emerging filtration technologies.

A new and ecological miniaturized method by spectrophotometry for quantification of vancomycin in dosage form

Vancomycin, an important antibiotic, is marketed as lyophilized powder. In the context of routine analysis of this product, the existence of a more advantageous and effective method is interesting. Thus, the objective of this work is to develop and validate a new analytical method, faster, low-cost, ecological and miniaturized for quantification of vancomycin in lyophilized powder using spectrophotometry in ultraviolet region. Buffer solution pH 6.8, quartz cuvette with capacity of 700 µL and 280 nm were chosen. The method proved to be linear in the range of 50-150 µg/mL (0.9997). The selectivity of the method was proven in two ways: The standard-sample overlay aimed to identify vancomycin in the sample; The forced degradation test (sample solutions prepared in 0.01 M HCl, 0.01 M NaOH and aqueous conditions and kept at 60 ºC by 8 hours, and UV 254 nm at ambient temperature during 24 hours) aimed to show the susceptibility of the method to consequently indicate the stability of the sample. It was precise in intraday (RSD 1.27%), interday (RSD 1.18%) and between analysts (RSD 1.92%) levels. It was robust when small variations were performed in seven important parameters (wavelength, cuvette, filtration step, dibasic and monobasic phosphate brand, ultrasound time and source of water). The accuracy was proved by the standard recovery test and showed mean recovery of 101.10%. This method can be applied in routine analysis of quality control of vancomycin lyophilized powder and it is an effective, accessible and ecological alternative, which follows the Green Analytical Chemistry principles, presenting less waste generation, no use of toxic solvents, smaller sample volumes and required diluents, which impacts on the final cost of the analyzes.

Investigation of an Intensified Thermo-Chemical Experimental Set-Up for Hydrogen Production from Biomass: Gasification Process Performance—Part I

Biomass gasification for energy purposes has several advantages, such as the mitigation of global warming and national energy independency. In the present work, the data from an innovative and intensified steam/oxygen biomass gasification process, integrating a gas filtration step directly inside the reactor, are presented. The produced gas at the outlet of the 1 MWth gasification pilot plant was analysed in terms of its main gaseous products (hydrogen, carbon monoxide, carbon dioxide, and methane) and contaminants. Experimental test sets were carried out at 0.25–0.28 Equivalence Ratio (ER), 0.4–0.5 Steam/Biomass (S/B), and 780–850 °C gasification temperature. Almond shells were selected as biomass feedstock and supplied to the reactor at approximately 120 and 150 kgdry/h. Based on the collected data, the in-vessel filtration system showed a dust removal efficiency higher than 99%-wt. A gas yield of 1.2 Nm3dry/kgdaf and a producer gas with a dry composition of 27–33%v H2, 23–29%v CO, 31–36%v CO2, 9–11%v CH4, and light hydrocarbons lower than 1%v were also observed. Correspondingly, a Low Heating Value (LHV) of 10.3–10.9 MJ/Nm3dry and a cold gas efficiency (CGE) up to 75% were estimated. Overall, the collected data allowed for the assessment of the preliminary performances of the intensified gasification process and provided the data to validate a simulative model developed through Aspen Plus software.

Effect of Pre-Heating Prior to Low Temperature 0.1 µm-Microfiltration of Milk on Casein–Whey Protein Fractionation

During skim milk microfiltration (nominal pore size of 0.1 µm) at 10 °C, the whey protein purity in the permeate is reduced by an enhanced serum casein permeation, primarily of β-casein. To decrease casein permeation, the possibility of a pre-heating step under pasteurization conditions before the filtration step was investigated, so as to shift the equilibrium from soluble serum casein monomers to impermeable micellar casein. Immediately after the pre-heating step, low temperature microfiltration at 10 °C was conducted before the casein monomers could diffuse into the serum. The hypothesis was that the dissociation of β-casein into the serum as a result of a decreasing temperature takes more time than the duration of the microfiltration process. It was found that pre-heating reduced the β-casein permeation during microfiltration without significantly affecting the flux and whey protein permeation, compared with a microfiltration at 10 °C without the pre-heating step. Furthermore, the addition of calcium (5 and 10 mM) not only reduced the casein permeation and thus increased the permeate purity, defined as a high whey protein-to-casein (g L−1/g L−1) ratio, but also decreased the filtration performance, possibly due to the structural alteration of the deposited casein micelle layer, rendering the deposit more compact and more retentive. Therefore, the possible combination of the addition of calcium and pre-heating prior to microfiltration was also investigated in order to evidence the potential increase of whey protein (WP) purity in the permeate in the case of Ca2+ addition prior to microfiltration. This study shows that pre-heating very close to low temperature microfiltration results in an increased purity of the whey protein fraction obtained in the permeate.

Platelet-rich plasma (PRP) in osteoarthritis (OA) knee: Correct dose critical for long term clinical efficacy

Despite encouraging results reported with regards to Platelet-rich plasma (PRP) application in osteoarthritis (OA) knee, still critical issues like conclusive structural evidence of its efficacy, standard dose and good manual method of preparation to obtain high yield remains unanswered. Present study is an attempt to optimise the dose and concentration of therapeutic PRP and its correlation with structural, physiologic efficacy with a new manual method of PRP preparation. A total of one hundred and fifty patients were randomized to receive either PRP (10 billion platelets) or hyaluronic acid (HA; 4 ml; 75 patients in each group) and followed up till 1 year. An addition of filtration step with 1 µm filter in manual PRP processing improved platelet recovery upto 90%. Significant improvements in WOMAC (51.94 ± 7.35 vs. 57.33 ± 8.92; P < 0.001), IKDC scores (62.8 ± 6.24 vs 52.7 ± 6.39; P < 0.001), 6-min pain free walking distance (+ 120 vs. + 4; P < 0.001) persisted in PRP compared to HA group at 1 year. Significant decline IL-6 and TNF-α levels observed in PRP group (P < 0.05) compared to HA at 1 month. Study demonstrated that an absolute count of 10 billion platelets is crucial in a PRP formulation to have long sustained chondroprotective effect upto one year in moderate knee OA.

Characterization of Used Lubricant Oil in a Latin-American Medium-Size City and Analysis of Options for Its Regeneration

Petroleum-derived products, such as lubricant oils, are non-renewable resources that, after use, must be collected and processed properly to avoid negative environmental impacts. A circular economy of used oils requires the re-refining and reuse of the same. Similar to most countries in Latin America, the management of used oils in Ecuador is still incipient and few cities collect and treat this material properly. In Cuenca, the ETAPA company collects ~1344 t/year of used oils, which are subjected to pretreatment operations prior to their use as fuel in a cement factory. However, combustion generates polluting gases and disallows the adding of value to the used oils. The lack of studies on the characterization and methods utilized for recovering used oils under the conditions found in medium-size Latin-American cities (e.g., Cuenca), alongside a lack of government policies, have hindered the adoption of re-refining operations. The objective of this work is to characterize the used lubricant oils in Cuenca, to compare them with the properties of used oils from other countries, and to suggest some re-refining technologies for oils with similar properties. Used oil samples were collected from mechanic shops and car-lubricating shops for characterization. Its physicochemical properties and metal contents are comparable to the used oils in other countries globally. Specifically, the flash point, kinematic viscosity, TBN, and concentrations of Zn, Cd, and Mg are similar to the properties of used oils in Iraq, Egypt, and the United Arab Emirates. Based on these results, the best re-refining option for used oils in Cuenca is extraction with solvents in which sedimentation and dehydration (already conducted in Cuenca) is followed by a solvent reaction process, a vacuum distillation process, a finishing process with bentonite, and a final filtration step.

Transfer of Lipophilic Drugs from Nanoemulsions into Lipid-Containing Alginate Microspheres

Knowledge about the release behavior and drug retention properties of colloidal carriers is of essential importance for quality control as well as to predict in vivo performance. When conducting release studies from such systems, the release media should preferentially contain lipophilic acceptor components in order to mimic physiological conditions. In this study, transfer from a trimyristin nanoemulsion into lipid-containing hydrogel beads was investigated for fenofibrate, cannabidiol, retinyl acetate, orlistat, and lumefantrine. To generate the acceptor system, a trimyristin nanoemulsion was incorporated into Ca-alginate microspheres (mean diameter ~40 µm) with a spraying method. Using this approach, the advantages of small lipophilic acceptor particles with a large interfacial area were combined with a single separation process from the donor via a filtration step. The method was applicable to distinguish between fast (fenofibrate) and slow drug transfer (lumefantrine) with good time resolution. Lipophilicity, estimated according to the calculated logP value of the respective drug, was a major factor influencing the transfer performance: the higher the logP value, the slower the transfer. This experimental setup is a promising technique to investigate the release of poorly water-soluble drugs from various types of nanocarriers under closer to physiological conditions than with many other methods currently applied.

Occupancy-collection models: Towards bias-corrected modeling of species' distributions using unstructured occurrence data from museums and herbaria

The digitization of museum collections as well as an explosion in citizen science initiatives has resulted in a wealth of data that can be useful for understanding the global distribution of biodiversity, provided that the well-documented biases inherent in unstructured opportunistic data are accounted for. While traditionally used to model imperfect detection using structured data from systematic surveys of wildlife, occupancy-detection models provide a framework for modelling the imperfect collection process that results in digital specimen data. In this study, we explore methods for adapting occupancy-detection models for use with biased opportunistic occurrence data from museum specimens and citizen science platforms using 7 species of Anacardiaceae in Florida as a case study. We explored two methods of incorporating information about collection effort to inform our uncertainty around species presence: (1) filtering the data to exclude collectors unlikely to collect the focal species and (2) incorporating collection covariates (collection type and history of previous detections) into a model of collection probability. We found that the best models incorporated both the background data filtration step as well as the incorporation of collector covariates associated with the probability of collection. We found that month, method of collection and whether a collector had previously collected the focal species were important predictors of collection probability. Efforts to standardize meta-data associated with data collection will improve efforts for modeling the spatial distribution of a variety of species.