scholarly journals Optimization of High-Throughput Multiplexed Phenotyping of Extracellular Vesicles Performed in 96-Well Microtiter Plates

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2368
Author(s):  
Malene Jørgensen ◽  
Jenni Sloth ◽  
Rikke Bæk
Keyword(s):  
Extracellular Vesicles ◽  
Room Temperature ◽  
Protein Microarray ◽  
Microarray Platform ◽  
Microtiter Plate ◽  
Storage Conditions ◽  
Array Analysis ◽  
Microtiter Plates ◽  
Associated Proteins ◽  
Technological Platform

Extracellular vesicles (EVs) are promising biomarkers for several diseases, however, no simple and robust methods exist to characterize EVs in a clinical setting. The EV Array analysis is based on a protein microarray platform, where antibodies are printed onto a solid surface that enables the capture of small EVs (sEVs) by their surface or surface-associated proteins. The EV Array analysis was transferred to an easily handled microtiter plate (MTP) format and a range of optimization experiments were performed within this study. The optimization was performed in a comprehensive analytical setup where the focus was on the selection of additives added to spotting-, blocking-, and incubation buffers as well as the storage of printed antibody arrays under different temperatures from one day to 12 weeks. After ending the analysis, the stability of the fluorescent signal was investigated at different storage conditions for up to eight weeks. The various parameters and conditions tested within this study were shown to have a high influence on each other. The reactivity of the spots was found to be preserved for up to 12 weeks when stored at room temperature and using blocking procedure IV in combination with trehalose in the spotting buffer. Similar preservation could be obtained using glycerol or sciSPOT D1 in the spotting buffers, but only if stored at 4 °C after blocking procedure I. Conclusively, it was found that immediate scanning of the MTPs after analysis was not critical if stored dried, in the dark, and at room temperature. The findings in this study highlight the necessity of performing optimization experiments when transferring an established analysis to a new technological platform.

scholarly journals Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2020 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2019 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

scholarly journals Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2019 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips. In particular, we took advantage of bradykinin-derived peptidic baits to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

scholarly journals Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

2020 ◽  
Author(s):  
Alessandro Gori ◽  
Alessandro Romanato ◽  
Greta Bergamaschi ◽  
Alessandro Strada ◽  
Paola Gagni ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Binding Capacity ◽  
Microarray Platform ◽  
Label Free ◽  
New Class ◽  
Associated Proteins ◽  
Packing Defects ◽  
Binding Peptides ◽  
On Chip

Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

scholarly journals Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Tobias Habicher ◽  
Tobias Klein ◽  
Jacqueline Becker ◽  
Andreas Daub ◽  
Jochen Büchs
Keyword(s):  
Controlled Release ◽  
Bacillus Licheniformis ◽  
Protease Activity ◽  
Microtiter Plate ◽  
Fed Batch ◽  
Batch Mode ◽  
Screening Process ◽  
Microtiter Plates ◽  
Batch Fermenter ◽  
Production Conditions

Abstract Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

scholarly journals Enhanced preservation of vacuum-packaged Atlantic salmon by hyperbaric storage at room temperature versus refrigeration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Liliana G. Fidalgo ◽  
Mário M. Q. Simões ◽  
Susana Casal ◽  
José A. Lopes-da-Silva ◽  
Ivonne Delgadillo ◽  
...  
Keyword(s):  
Atlantic Salmon ◽  
Microbial Activity ◽  
Room Temperature ◽  
Energy Savings ◽  
Textural Properties ◽  
Storage Conditions ◽  
Volatile Profile ◽  
Fragmentation Index ◽  
C 30 ◽  
Water Holding

AbstractHyperbaric storage at room temperature (HS/RT: 75 MPa/25 °C) of vacuum-packaged fresh Atlantic salmon (Salmo salar) loins was studied for 30 days and compared to atmospheric pressure at refrigerated temperatures (AP/5 °C, 30 days) and RT (AP/25 °C, 5 days). Most of the fatty acids were not affected by storage conditions, with only a slight decrease of docosahexaenoic acid (DHA) content (n-3 polyunsaturated fatty acid) for AP samples, reflected in the lower polyene index values obtained and higher oxidation extent. For HS, a lower lipid oxidation extension and a slower increase of myofibrillar fragmentation index values were observed, when compared to AP samples. The volatile profile was similar for the HS and fresh samples, with the HS samples retaining fresh-like alcohols and aldehydes components, which disappeared in AP samples, mainly in AP/25 °C samples. The volatile profile for AP samples (5 and 25 °C) revealed mostly spoilage-like compounds due to microbial activity. Drip loss increased progressively during the 30 days of storage under HS, while a slight decrease of water holding capacity after 5 days was observed, increasing further after 30 days. Regarding textural properties, only resilience was affected by HS, decreasing after 30 days. So, HS/RT could represent an interesting extended preservation methodology of fresh salmon loins, since allows retaining important physicochemical properties for at least 15 days, while refrigeration after 5 days showed already volatile spoilage-like compounds due to microbial activity. Furthermore, this methodology allows additional considerable energy savings when compared to refrigeration.

scholarly journals The holding temperature of blood during a delay to processing can affect serum and plasma protein measurements

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Milton Ashworth ◽  
Benjamin Small ◽  
Lucy Oldfield ◽  
Anthony Evans ◽  
William Greenhalf ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Room Temperature ◽  
Serum Levels ◽  
Data Interpretation ◽  
Blood Samples ◽  
Specific Manner ◽  
Associated Proteins ◽  
Holding Temperature ◽  
Pai 1 ◽  
Meaningful Data

AbstractAccurate blood-borne biomarkers are sought for diagnosis, prognosis and treatment stratification. Consistent handling of blood is essential for meaningful data interpretation, however, delays during processing are occasionally unavoidable. We investigated the effects of immediately placing blood samples on ice versus room temperature for 1 h (reference protocol), and holding samples on ice versus room temperature during a 3 h delay to processing. Using Luminex multi-plex assays to assess cytokines (n = 29) and diabetes-associated proteins (n = 15) in healthy subjects, we observed that placing blood samples immediately on ice decreased the serum levels of several cytokines, including PAI-1, MIP1-β, IL-9, RANTES and IL-8. During a delay to processing, some analytes, e.g. leptin and insulin, showed little change in serum or plasma values. However, for approximately half of the analytes studied, a delay, regardless of the holding temperature, altered the measured levels compared to the reference protocol. Effects differed between serum and plasma and for some analytes the direction of change in level varied across individuals. The optimal holding temperature for samples during a delay was analyte-specific. In conclusion, deviations from protocol can lead to significant changes in blood analyte levels. Where possible, protocols for blood handling should be pre-determined in an analyte-specific manner.

scholarly journals Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Mario Codemo ◽  
Sandra Muschiol ◽  
Federico Iovino ◽  
Priyanka Nannapaneni ◽  
Laura Plant ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Plasma Membrane ◽  
Streptococcus Pneumoniae ◽  
Extracellular Vesicles ◽  
Factor H ◽  
Host Cells ◽  
Immunomodulatory Effects ◽  
Content Type ◽  
Associated Proteins ◽  
Tract Infections

ABSTRACTGram-positive bacteria, including the major respiratory pathogenStreptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.IMPORTANCEStreptococcus pneumoniaeis a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

Characteristics of Packaged Water under Different Storage Conditions Within Osogbo Metropolis

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
O.O Fadipe
Keyword(s):  
Room Temperature ◽  
Control Unit ◽  
Storage Conditions ◽  
Prolonged Storage ◽  
E Coli ◽  
Physico Chemical ◽  
Routine Basis ◽  
Regulatory Bodies ◽  
Microbiological Analyses ◽  
Sachet Water

The study investigated the characteristics of packaged water stored under ambient and sunlight conditions. This is with a view to testing the effect of prolonged storage under different storage conditions on its quality. In addition it analyzed the interactions between the parameters. Two packs each of bottled and sachet water was purchased from each factory at the point of production and ready for distribution to wholesalers. Twenty eight pieces of packaged water from each factory were kept at room temperature and the same quantity were kept under sunlight. Physico-chemical and microbiological analyses were carried out on the remaining packaged water within 24hrs. Half of the samples stored at the two storage conditions were removed for analysis at 3 weeks while the remaining half was analyzed after 6weeks. The physico-chemical characteristics were within the WHO recommended values except for the pH of some samples that have values in the acidic range of 6.2-6.48. All the physico-chemical values increased for samples kept under sunlight. All the water samples showed growth in faecal coliform (4-46 cfu/100 mL) and E. coli (0-13 cfu/100 mL) for samples kept under sunlight at three weeks and this growth increased to the sixth week. The presence of E. coli is an indication that the packaged water is not pure. Displaying packaged water under the sunlight and storing beyond 3 weeks by vendors have effect on the potability of the product. The regulatory bodies should raise awareness and ensure manufacturer have a quality control unit to test on a routine basis.

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