Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60–140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

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Membrane technology has emerged as a leading tool worldwide for effective CO 2 separation because of its well-known advantages, including high surface area, compact design, ease of maintenance, environmentally friendly nature, and costeffectiveness. Polymeric and inorganic membranes are generally utilized for the separation of gas mixtures. The mixed-matrix membrane (MMM) utilizes the advantages of both polymeric and inorganic membranes to surpass the trade-off limits. The high permeability and selectivity of MMMs by incorporating different types of fillers exhibit the best performance for CO 2 separation from natural gas and other flue gases. The recent progress made in the field of MMMs having different types of fillers is emphasized. Specifically, CO 2 /CH 4 and CO 2 /N 2 separation from various types of MMMs are comprehensively reviewed that are closely relevant to natural gas purification and compositional flue gas treatment

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This paper presents the first report of anthelmintic resistance (AR) in dairy goats in a desert (Pakistan). Three breeds of dairy goats, i.e. Dera Din Panah, Pak Angora and Beetal, kept at Government Livestock Farm, Rakh Khairewala, district Jhang/Layya, Pakistan, were surveyed for gastrointestinal nematodes (GINs) resistant to commonly used three anthelmintics, i.e. benzimidazole, levamisole and ivermectin. Sixty animals of each breed were selected randomly on the basis of their weight and egg count. Three commonly used anthelmintics, viz., oxfendazole (three different preparations of oxfendazole: fendamex, oxazole, systamex), levamisole and ivermectin, were given at the recommended dose to five groups while one untreated group was kept as control for each breed. Faecal egg counts, faecal egg count reduction test, postmortem worm count and copro-culture were performed to assess the efficacy of selected anthelmintics. The prevalent species of GINs exhibited resistance against all three preparations of oxfendazole. Levamisole in two breeds and ivermectin in all the breeds led to reduction (P< or =0.05) of prevalent species of GINs in both flocks. Haemonchus controtus and Trichostrongylus species exhibited the presence of resistance against oxfendazole preparations which exhibited low efficacy (P> or =0.05). The farm management practices along with the results of the present study revealed the presence of multiple anthelmintic resistant GINs of dairy goats kept in a desolated tract.

Both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) are rapidly overcoming the current array of drugs. One hundred and fifty isolates from a hospital were studied for resistance towards linezolid and vancomycin. Fifty-four (36.0 %) isolates were MRSA. Both MRSA and MSSA showed high resistance towards linezolid when using the disc diffusion method, with the figures being 48.1 and 29.2 %, respectively. The figures for the E-test were 46.3 and 27.0 %, respectively. The vancomycin resistance was remarkable in MRSA (14.8 %), but relatively low in MSSA (3.1 %). The E-test results were 13.0 and 4.16 %, respectively. The cfr gene was detected in 78 % of linezolid-resistant isolates and the vanA operon was detected in 74 % of vancomycin-resistant isolates. This level of resistance against linezolid and vancomycin is unprecedented. These results are alarming and highlight the threat of non-treatable S. aureus strains.