Sisäilmastoseminaari 2019

MALDI-TOF massaspektrometrialla voidaan luokitella ja tunnistaa mikro-organismeja niiden proteiiniprofiilin mukaan. Tämä kustannustehokas ja nopea menetelmä on otettu laajasti käyttöön mikrobiologisessa analytiikassa. Artikkelissa kuvataan aloitettua selvitys­työtä menetelmän hyödynnettävyydestä asumisterveystutkimuksissa. Esikokeissa menetelmää testattiin 10 Aspergillus-kannalla sekä 50 asumisterveysnäytteistä eristetyllä aktinomykeettikannalla. Aspergillus-lajien tunnistus onnistui tyydyttävästi. Aktinomykeetit tunnistettiin käytetyn tietokannan suppeuden vuoksi pääosin vain sukutasolle. Menetelmän käyttöönotto edellyttäisi MALDI-TOF MS kirjastotietokantojen laajentamista DNA-sekvensoinnilla tunnistetuilla ympäristömikrobikannoilla</p

Enrichment of bovine milk-derived extracellular vesicles using surface-functionalized cellulose nanofibers

The isolation of extracellular vesicles (EVs) from milk, a complex mixture of colloidal structures having a comparable size to EVs, is challenging. Although ultracentrifugation (UC) has been widely used for EV isolation, this has significant limitations, including a long processing time at high g-force conditions and large sample volume requirements. We introduced a new approach based on nature nanoentities cellulose nanofibers (CNFs) and short time and low g-force centrifugation to isolate EVs from various milk fractions. The flexible and entangled network of CNFs forms nanoporous, which entraps the EVs. Further, positively charged CNFs interact with anionic EVs through an electrostatic attraction, promoting their isolation with efficiency comparable with UC. The functionality and toxicity of isolated milk EVs were tested in Caco2 cells. Overall, the newly developed approach provides straightforward isolation and biocompatibility and preserves the natural properties of the isolated EVs, enabling further applications

Isolation and characterization of exosomes from bovine milk

Exosomes are extracellular vesicles of endosomal origin with a diameter of 30-150 nms that contribute to cell to cell communication and may be involved in interspecies communication on the nano-level. Exosomes contain wide array of bioactive components from micro RNAs to proteins and bioactive lipids. Milk is a unique platform of information from mother to infant in mammals with unique content of growth factors and immunogens. Some of these messages of biological programming can be transferred via exosomes. Bovine milk contains vesicles of all sizes in very high concentrations. The abundance of vesicles in milk provides a platform for commercial scale purification of bioavailable extracellular vesicles, including exosomes. Isolation of exosomes from such complex media as milk provides challenges with high fat and protein content with similar physiochemical properties as milk exosomes. Here we demonstrate an efficient protocol for isolation of milk exosomes with the use of differential centrifugation followed by secondary purification with size exclusion chromatography or sucrose gradient centrifugation. Resulting samples were analysed for exosome enriched proteins with western blot for ALIX, TSG101 and CD81. Further analysis was done with mass spectrometry to identify the complete protein profile of isolated vesicles that included also exosomal marker proteins HSC70, CD81 and CD9

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

202

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance

Abstract Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50–150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70–500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10–70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with ¹²⁹Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool

APOE Genotype Disclosure and Lifestyle Advice in a Randomized Intervention Study with Finnish Participants

202

Enrichment of bovine milk-derived extracellular vesicles using surface-functionalized cellulose nanofibers

Abstract The isolation of extracellular vesicles (EVs) from milk, a complex mixture of colloidal structures having a comparable size to EVs, is challenging. Although ultracentrifugation (UC) has been widely used for EV isolation, this has significant limitations, including a long processing time at high g-force conditions and large sample volume requirements. We introduced a new approach based on nature nanoentities cellulose nanofibers (CNFs) and short time and low g-force centrifugation to isolate EVs from various milk fractions. The flexible and entangled network of CNFs forms nanoporous, which entraps the EVs. Further, positively charged CNFs interact with anionic EVs through an electrostatic attraction, promoting their isolation with efficiency comparable with UC. The functionality and toxicity of isolated milk EVs were tested in Caco2 cells. Overall, the newly developed approach provides straightforward isolation and biocompatibility and preserves the natural properties of the isolated EVs, enabling further applications