Exosomes, derived from stem cells, have great promise in regenerative medicine due to their capabilities of ameliorating inflammation, preventing tissue damage and promoting healing, which in part are associated with the exosomal RNA/miRNA. The application of mesenchymal stem cell exosomes in treating hepatic disorders including nonalcoholic fatty liver disease has drawn much attention. In this chapter, we describe our experience in culturing human mesenchymal stem cells and isolating their exosomes from culture medium through ultracentrifugation. Methods to extract exosomal RNA/miRNA are also discussed.
- MeSH
- Exosomes * genetics MeSH
- Stem Cells MeSH
- Humans MeSH
- Mesenchymal Stem Cells * MeSH
- MicroRNAs * genetics MeSH
- Ultracentrifugation MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using 15N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using 15N.
- MeSH
- RNA, Bacterial chemistry genetics isolation & purification metabolism MeSH
- Nitrogen-Fixing Bacteria genetics metabolism MeSH
- Centrifugation, Density Gradient MeSH
- DNA, Bacterial chemistry genetics isolation & purification metabolism MeSH
- Nitrogen Fixation genetics physiology MeSH
- Isotope Labeling methods MeSH
- Nitrogen Isotopes metabolism MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
- MeSH
- Anisotropy MeSH
- Cell Membrane chemistry MeSH
- Cholesterol chemistry MeSH
- Detergents chemistry MeSH
- Chromatography, Gel MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Lipid Bilayers chemistry MeSH
- Lipids chemistry MeSH
- Maleates chemistry MeSH
- Fatty Acids chemistry MeSH
- Membrane Microdomains chemistry MeSH
- Membrane Proteins chemistry MeSH
- Membranes, Artificial MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Polymers chemistry MeSH
- Scattering, Radiation MeSH
- Solubility MeSH
- Styrene chemistry MeSH
- Light MeSH
- T-Lymphocytes cytology MeSH
- Ultracentrifugation MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- FOLANDROL,
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Fertilization in Vitro methods MeSH
- Sperm Injections, Intracytoplasmic methods MeSH
- Humans MeSH
- Magnetics MeSH
- Infertility, Male complications MeSH
- Oligospermia drug therapy MeSH
- Dietary Supplements MeSH
- Cell Separation methods MeSH
- Spermatozoa cytology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Cryopreservation methods MeSH
- Sperm Motility physiology MeSH
- Silicon Dioxide chemistry MeSH
- Povidone chemistry MeSH
- Proteomics MeSH
- Fishes physiology MeSH
- Spermatozoa physiology MeSH
- Semen Preservation methods MeSH
- Cell Survival physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Next-generation sequencing has opened avenues to studying complex populations such as the bacteriome (all bacteria), mycobiome (all fungi), and virome (all viruses in a given sample). Viromes are less often investigated as compared to bacteriomes. The reasons are mostly methodological: because no common pan-viral sequence signature exists, metagenomic sequencing remains the only option. This brings about the need of laborious virus enrichment, multiple signal amplification steps with virtually no possibility of interim quality control, and complicated bioinformatic analysis of the ensuing sequence data. Nevertheless, over the past decade virome sequencing has been enormously successful in identifying new agents in human and animal diseases, and in characterizing viruses in various ecological niches. Recently, virome sequencing has been also employed in studies of non-infectious diseases, which has brought about new challenges of sensitivity, costs, and reproducibility in testing of large sets of samples. Here, we present a detailed protocol that has been utilized in virome studies where hundreds of samples had to be reliably tested in order to assess the association of the stool virome with susceptibility to type 1 diabetes, a non-infectious autoimmune disease.
- MeSH
- Bacteriophages classification genetics isolation & purification MeSH
- DNA, Viral genetics isolation & purification MeSH
- Feces virology MeSH
- Gene Library MeSH
- Metagenome * MeSH
- Metagenomics * methods MeSH
- Polymerase Chain Reaction MeSH
- RNA, Viral genetics isolation & purification MeSH
- Gastrointestinal Microbiome * MeSH
- Ultracentrifugation MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Importance: Recent studies have shown that Friedewald underestimates low-density lipoprotein cholesterol (LDL-C) at lower levels, which could result in undertreatment of high-risk patients. A novel method (Martin/Hopkins) using a patient-specific conversion factor provides more accurate LDL-C levels. However, this method has not been tested in proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor-treated patients. Objective: To investigate accuracy of 2 different methods for estimating LDL-C levels (Martin/Hopkins and Friedewald) compared with gold standard preparative ultracentrifugation (PUC) in patients with low LDL-C levels in the Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Patients With Elevated Risk (FOURIER) trial. Design, Setting, and Participants: The FOURIER trial was a randomized clinical trial of evolocumab vs placebo added to statin therapy in 27 564 patients with stable atherosclerotic cardiovascular disease. The patients' LDL-C levels were assessed at baseline, 4 weeks, 12 weeks, 24 weeks, and every 24 weeks thereafter, and measured directly by PUC when the level was less than 40 mg/dL per the Friedewald method (calculated as non-HDL-C level - triglycerides/5). In the Martin/Hopkins method, patient-specific ratios of triglycerides to very low-density lipoprotein cholesterol (VLDL-C) ratios were determined and used to estimate VLDL-C, which was subtracted from the non-HDL-C level to obtain the LDL-C level. Main Outcomes and Measures: Low-density lipoprotein cholesterol calculated by the Friedewald and Martin/Hopkins methods, with PUC as the reference method. Results: For this analysis, the mean (SD) age was 62.7 (9.0) years; 2885 of the 12 742 patients were women (22.6%). A total of 56 624 observations from 12 742 patients had Friedewald, Martin/Hopkins, and PUC LDL-C measurements. The median difference from PUC LDL-C levels for Martin/Hopkins LDL-C levels was -2 mg/dL (interquartile range [IQR], -4 to 1 mg/dL) and for Friedewald LDL-C levels was -4 mg/dL (IQR, -8 to -1 mg/dL; P < .001). Overall, 22.9% of Martin/Hopkins LDL-C values were more than 5 mg/dL different than PUC values, and 2.6% were more than 10 mg/dL different than PUC levels. These were significantly less than respective proportions with Friedewald estimation (40.1% and 13.3%; P < .001), mainly because of underestimation by the Friedewald method. The correlation with PUC LDL-C was significantly higher for Martin/Hopkins vs Friedewald (ρ, 0.918 [95% CI 0.916-0.919] vs ρ, 0.867 [0.865-0.869], P < .001). Conclusions and Relevance: In patients achieving low LDL-C with PCSK9 inhibition, the Martin/Hopkins method for LDL-C estimation more closely approximates gold standard PUC than Friedewald estimation does. The Martin/Hopkins method may prevent undertreatment because of LDL-C underestimation by the Friedewald method. Trial Registration: ClinicalTrials.gov Identifier: NCT01764633.
- MeSH
- Anticholesteremic Agents therapeutic use MeSH
- Atherosclerosis blood drug therapy MeSH
- Cholesterol, HDL analysis blood MeSH
- Risk Assessment MeSH
- Antibodies, Monoclonal, Humanized MeSH
- Hyperlipidemias blood drug therapy MeSH
- Cholesterol, LDL analysis blood MeSH
- Middle Aged MeSH
- Humans MeSH
- Antibodies, Monoclonal therapeutic use MeSH
- Randomized Controlled Trials as Topic MeSH
- Aged MeSH
- Statistics as Topic methods MeSH
- Triglycerides analysis blood MeSH
- Ultracentrifugation methods MeSH
- Cholesterol, VLDL analysis blood MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Blastocystis is a common inhabitant of the human gut, colonizing at least one billion people at a prevalence ranging from <10% to 100% in healthy human populations globally. The majority of carriers remain asymptomatic, suggesting that Blastocystis is largely a commensal, though Blastocystis has also been implicated in disease in some people. However, there are no in vivo model systems in which to experimentally test the impact of Blastocystis on mammalian hosts and the gut ecosystem and determine which factors underlie these variable clinical outcomes. We evaluated a rat model for sustaining of a human-derived Blastocystis ST1 and assess colonization success and longevity. Because of the broad host range of Blastocystis, we compared the rat with three other rodent species to establish the reproducibility of our method. Blastocystis was introduced by esophageal gavage and colonization success evaluated by Blastocystis culture. Culture was also used to determine that all animals were negative prior to colonization and negative controls remain Blastocystis-free. In this study, Blastocystis ST1 established in 100% of the outbred rats (Rattus norvegicus) and gerbils (Meriones unguiculatus) challenged. Rats were colonized asymptomatically for more than one year, but Blastocystis ST1 was not transmitted between rats. Mus musculus strain CD1 and Mastomys coucha were not susceptible to Blastocystis ST1. Thus, rats appear to be a suitable in vivo model for studies of Blastocystis ST1, as do gerbils though testing was less extensive. This work lays the foundation for experimental work on the role of Blastocystis in health and disease.
- MeSH
- Blastocystis growth & development pathogenicity MeSH
- Blastocystis Infections diagnosis parasitology MeSH
- Centrifugation, Density Gradient MeSH
- Feces parasitology MeSH
- Gerbillinae MeSH
- Rats MeSH
- Humans MeSH
- Disease Models, Animal * MeSH
- Murinae MeSH
- Mice MeSH
- Disease Susceptibility MeSH
- Specific Pathogen-Free Organisms MeSH
- Rats, Wistar MeSH
- Health Status MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Comparative Study MeSH
The opportunistic Gram-negative bacterium Burkholderia cenocepacia causes lethal infections in cystic fibrosis patients. Multivalent mannoside derivatives were prepared as potential inhibitors of lectin BC2L-A, one of the virulence factors deployed by B. cenocepacia in the infection process. An (α1→2)-thio-linked mannobioside mimic bearing an azide functionalized aglycon was conjugated to different multivalent scaffolds such as propargylated calix[4]arenes, methyl gallate and pentaerythritol by azide-alkyne 1,3-dipolar cycloaddition. The interaction between the glycoclusters and the mannose binding BC2L-A lectin from B. cenocepacia was examined by isothermal microcalorimetry, surface plasmon resonance, inhibition of yeast agglutination and analytical ultracentrifugation.
- MeSH
- Agglutination Tests MeSH
- Burkholderia cenocepacia chemistry MeSH
- Calorimetry methods MeSH
- Yeasts drug effects MeSH
- Mannose-Binding Lectin chemistry metabolism pharmacology MeSH
- Ligands MeSH
- Mannosides chemical synthesis chemistry metabolism MeSH
- Surface Plasmon Resonance MeSH
- Chemistry Techniques, Synthetic MeSH
- Ultracentrifugation methods MeSH
- Publication type
- Journal Article MeSH
Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.
