Use Of Density Gradients Research Articles

Nanopore Direct RNA Sequencing of Monosome- and Polysome-Bound RNA.

Polysome fractionation makes use of density gradients and ultracentrifugation to separate transcripts based on their specific number of bound ribosomes, and can be combined with downstream analysis such as cDNA-seq (commonly known as RNA-seq), microarray analysis, RT-qPCR, or Northern blotting. Here, we describe the application of Nanopore direct RNA sequencing to quantify monosome- and polysome-bound full-length transcripts after polysome fractionation, RNA cleanup, and size selection, using the yeast glucose stress response as an example use case.

251 FACTORS INFLUENCING THE SUCCESS OF EQUINE INTRACYTOPLASMIC SPERM INJECTION IN A CLINICAL PROGRAM

Intracytoplasmic sperm injection (ICSI) is currently being used clinically in horses, but little information is available on factors affecting its success. We have conducted research trials and evaluated data from our clinical ICSI caseload, currently over 450 cases per year, to provide information in this area. In the following summaries, blastocyst data are reported per injected oocyte; clinical data are from 2013 and 2014. Clinically, when immature follicles were aspirated, the number of follicles aspirated per mare decreased significantly with mare age, from 16.2 at age 12–15 to 8.9 at age 24–25; however, maturation and blastocyst rates of recovered oocytes did not differ significantly. Immature oocytes shipped to the laboratory by referring veterinarians yielded a significantly higher blastocyst rate than that for immature oocytes aspirated at the laboratory (27 v. 21%, respectively). Shipped oocytes recovered from stimulated preovulatory follicles yielded a higher blastocyst rate per oocyte than did shipped immature oocytes (39 v. 27%, respectively), but provided fewer mature oocytes per aspiration (1.0 v. 4.5 for immature). From research data, administration of gentamicin and ampicillin to mares before immature oocyte aspiration did not affect blastocyst rate. Holding the aspirate for ~1.5 h at ambient temperature (26 to 33°C) was associated with a blastocyst rate of 32%; however, holding for 2 h at 32°C yielded only 16% blastocysts v. 23% for control. Blastocysts (18%) were obtained from oocytes recovered in the nonbreeding season (December and January). Holding oocytes at room temperature overnight before maturation did not affect blastocyst rate (25 to 34%), nor did inclusion of zinc in the maturation medium (18 to 31%). Diluting and refreezing semen to increase doses available for ICSI did not affect blastocyst rate (23 to 27%); blastocysts (13%) were also obtained after injection of nonmotile sperm. Significant differences in cleavage and blastocyst rates were identified among stallions. For one stallion, use of density gradient plus swim-up before ICSI increased cleavage (49 v. 18%) and blastocyst rates (11 v. 0%) compared to density gradient alone. Blastocyst production was not affected by the amount of glucose added to a human embryo culture medium (0 or 5 mM added glucose on Days 0–5, then 10 or 20 mM added glucose; 31 to 46% blastocysts). Replacement of 10% FBS in embryo culture medium with a mixture of FBS, human serum replacement, and equine follicular fluid lowered blastocyst rate (15 v. 37% for FBS alone). Clinically, embryos were vitrified or were shipped to embryo transfer centers for transfer. There was no significant difference in ongoing pregnancy rate for embryos from shipped immature v. preovulatory oocytes (54 and 69%). For immature oocytes, embryos developing to blastocyst on Day 10 or 11 had a lower ongoing pregnancy rate after transfer (40 and 0%) than did those developing on Days 7 to 9 (51 to 75%). This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by the Clinical Equine ICSI Program, Texas A&M University.

Comparison of tissue sample processing methods for harvesting the viral metagenome and a snapshot of the RNA viral community in a turkey gut

RNA viruses have been associated with enteritis in poultry and have been isolated from diseased birds. The same viral agents have also been detected in healthy flocks bringing into question their role in health and disease. In order to understand better eukaryotic viruses in the gut, this project focused on evaluating alternative methods to purify and concentrate viral particles, which do not involve the use of density gradients, for generating viral metagenome data. In this study, the sequence outcomes of three tissue processing methods have been evaluated and a data analysis pipeline has been established for RNA viruses from the gastrointestinal tract. In addition, with the use of the best method and increased sequencing depth, a glimpse of the RNA viral community in the gastrointestinal tract of a clinically normal 5-week old turkey is presented. The viruses from the Reoviridae and Astroviridae families together accounted for 76.3% of total viruses identified. The rarefaction curve at the species level further indicated that majority of the species diversity was included with the increased sequencing depth, implying that viruses from other viral families were present in very low abundance.

Modified SLAU2 scheme with enhanced shock stability

Two alternate modifications are proposed to the dissipation term in the mass flux computation of the low dissipation AUSM scheme (SLAU2) developed recently by Kitamura and Shima [1,2]. These modifications are required to remove the odd–even type instability that results in lateral oscillations behind oblique shocks predicted by MUSCL based higher order versions of SLAU2. The first modification involves switching between the original term in SLAU2 and one similar to corresponding term in AUSM+-up. The second modification involves use of density gradient aligned velocity instead of total velocity in SLAU2 (or face normal velocity as in AUSM+-up) in calculation of Mach number that is required for computing this term. It is observed that the second alternative not only delivers better results but also has a more easily differentiable numerical flux that enables easier implicit computations while not altering the simplicity of original SLAU2. The method also renders SLAU2 with a good balance between shock stability and contact capturing ability.

Preparation of biologically active subcellular fractions using the Balch homogenizer

Obtaining vesicular fractions from cell lines or animal tissue is both time and technically intensive. The presence of plasma membrane and nuclear contaminants within a preparation is often dependent on the method of homogenization and is usually mitigated through the use of density gradients. We have developed a method that utilizes Balch homogenization and differential centrifugation to obtain two distinct vesicular fractions along with purified nuclear, cytoplasmic, and ghost fractions within a 3-h period of time without the use of density gradients. Importantly, these fractions maintain their biologic activity following isolation and may be used for both localization and biochemical analyses.

Advances in the Characterization of Particle Size Distributions of Abrasive Particles used in CMP

ABSTRACTThe use of density gradient stabilized centrifugal sedimentation (disc centrifugation) for the characterization of abrasive particles in CMP slurries is reported. For slurries prepared with ceria and colloidal silica abrasives, the technique is demonstrated as capable of providing highly repeatable analyses of the abrasive's apparent particle size distribution (PSD). The addition of water soluble organic additives to the slurries is shown to produce large shifts in the apparent PSD relative to the PSD of the pure abrasive particles. Particle-additive interactions driving the shift in apparent PSD could not be interpreted with confidence due to the lack of accurate densities for particle-abrasive complexes formed in the slurry. To address this problem, sucrose density gradients prepared using H2O and D2O were tested as spin fluids of different densities to analyze a colloidal silica standard with a narrow PSD. Preliminary results comparing experimental disc centrifuge data with theoretical simulations of the disc sedimentation pattern suggest that this technique can potentially characterize both the particle diameter and density of abrasive particles in a CMP slurry.

Eosinophil Apoptosis Induced by Fungal Immunomodulatory Peptide- fve via Reducing IL-5α Receptor

Eosinophils are important effector cells in the pathogenesis of allergic bronchial asthma. Enhancement of eosinophil apoptosis has been considered to have therapeutic effect on allergic disease. Fungal immunomodulatory peptide (FIP)-fve has been reported to possess immunoprophylactic activities for allergic diseases. The purpose of this study was to investigate the modulation of FIP-fve on human eosinophil survival derived from allergic asthmatic patients. Eosinophils were obtained from allergic asthmatic patients and purified with the use of density gradients and immunomagnetic beads negative selection. Apoptosis was assessed by annexin V and propidium iodide. The apoptotic signal protein, CD95 and IL-5 receptor expression were assessed by Western blot and flow cytometric analysis. When the eosinophils were treated with FIP-fve in the presence of IL-5, IL-5-enhanced eosinophil survival diminished. FIP-fve could reduce IL-5-mediated survival of eosinophils and decrease IL-5Ralpha expression. In the presence of FIP-fve, CD95 expression was upregulated and Bcl-xL and pro-caspase 3 expression were downregulated in cultured eosinophils. The results suggest that FIP-fve can inhibit IL-5-mediated survival of eosinophils through the modulation of cytokine receptor expression and apoptotic signal protein production. The modulatory effect of FIP-fve on eosinophil apoptosis in vitro indicates that it may have some therapeutic effect on eosinophil-related allergic inflammation in vivo.

Isolation of human eosinophils from peripheral blood using hypotonic lysis and centrifugation

We describe an alternative method for rapidly purifying viable and structurally intact eosinophils from human peripheral blood using controlled hypotonic lysis and centrifugation. This method has significant advantages in that it does not require the use of density gradients, antibodies, magnetic beads, or ammonium chloride lysis procedures.

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44 + cells, including up to 67% CMRF-44 + CD14 − CD19 − DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44 + cells (range 19–99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

Ligation of CD45 and the isoforms CD45RA and CD45RB accelerates the rate of constitutive apoptosis in human eosinophils

Background: Eosinophils are important effector cells in asthma pathogenesis, and an understanding of the mechanisms involved in eosinophil apoptosis induction might thus be relevant to the resolution of asthmatic inflammation. Objective: Our aim was to determine the role of the common leukocyte antigen CD45 and the isoforms CD45RA, CD45RB, and CD45RO in human eosinophil apoptosis induction. Methods: Immmunostaining and flow cytometry were used to assess CD45 and CD45 isoform expression by eosinophils purified with use of density gradients and immunomagnetic negative selection. Apoptosis induction was measured by binding of fluorescein isothiocyanate–labeled annexin V to eosinophils cultured for 20 hours alone or with saturating quantities of mAb against CD45, CD45RA, CD45RB, CD45RO, CD9, CD11b, and isotype-matched controls in the presence or absence of GM-CSF. Results: Freshly isolated eosinophils had high expression of CD45 and CD45RO, modest expression of CD45RB, and low expression of CD45RA. Eosinophils cultured alone for 20 hours were found to be approximately 20% to 25% apoptotic. Incubation with mAb against CD45, CD45RA, and CD45RB resulted in significant ( P < .005) enhancement (>100%) of their constitutive rate of apoptosis. Incubation with CD45RO, CD11b, CD9 mAb, or isotype controls had no significant effect on the rate of eosinophil constitutive apoptosis. The addition of GM-CSF inhibited eosinophil apoptosis but did not prevent CD45, CD45RA, or CD45RB mAb-dependent apoptosis induction. Conclusion: These data indicate that ligation of CD45, CD45RA, or CD45RB represents a novel pathway for the induction of apoptosis in human eosinophils. (J Allergy Clin Immunol 1999;104:1244-50.)

LDL particle size: an important drug target?

Low density lipoprotein (LDL) is the main carrier of plasma cholesterol and a major component of atherosclerotic plaque [1]. Lowering LDL cholesterol reduces coronary events and mortality from coronary artery disease (CAD) [2–4], however, the relation between LDL cholesterol concentration and (CAD) is complex. Many patients with CAD have plasma LDL cholesterol concentrations in the normal rangefor the general population [5]. Thus, it could be that coronary risk goes beyond LDL cholesterol concentration to the characteristics of the LDL particles themselves. The purpose of this communication is to address the issue of whether LDL particle size and density influences its atherogenecity and how this might be modified by drug therapy.

Polystyrene latex fractionation and particle size determination: The use of density gradients in a low-speed centrifuge

The technique of low-speed centrifugation in density gradients can be used to determine polystyrene latex particle diameters. Particles in the 0.1–0.5 μm diameter range were successfully analyzed, using Ficoll density gradients generated by osmocentrifugation and zonal centrifugation speeds of 3000 rpm. Using this technique, the supernatant of an aged, coagulated latex (nominal diameter 109 nm) was fractionated in six bands, corresponding to singlets, doublets and up to hexaplets. Using another latex, to which NaCl was added to give various salt concentrations (above and below the critical coagulation concentration), latex zonal centrifugation revealed various bands, even at [NaCl]=5 × 10 −5 M. A turbidimetric scan of cell contents, in samples containing NaCl, showed that there are significant particle concentrations in the spaces between successive bands; this may be due to particle-particle reversible association or to aggregate sedimentation coefficient polydispersity, arising from geometrical (shape, interparticle distance) changes, among aggregates.

Harvesting of Scenedesmus obliquus in wastewaters: Auto‐ or bioflocculation?

Autoflocculation and bioflocculation are considered to be the most promising means for the economical harvesting of microalgae. We have therefore studied these phenomena with cultures of Scenedesmus obliquus produced during biological tertiary wastewater treatment. The quantity of extracellular polymers produced during ageing of the cultures proved insufficient to initiate bio-flocculation while the concentration of Ca(2+) and PO(4) (3-) of the treated effluent were too low to induce autoflocculation. It has been shown, however, that the algae sediment more readily upon ageing, possibly as a result of increased cell density. The use of density gradients made with Percoll (a colloidal solution of silica particles) allowed measurement of the true cell density and showed that this increases when cultures enter the declining growth phase. The quality of the biomass thus harvested is, however, considerably impaired, protein content decreasing from 62.7% (dry wt) during the exponential growth phase (day 5) to 14% at the end of cultures (day 21).

Measurement of cell-mediated immunity against bovine papilloma virus by lymphoproliferative reactions

To study cellular immunity towards bovine papillomavirus (BPV), calves were infected intradermally with BPV-1, and the cellular immune response was measured by the lymphocyte proliferation assay. Peripheral blood lymphocytes were obtained which in control experiments were highly reactive towards mitogen stimulation. Different batches of BPV-1 were prepared by the use of density gradients. In the first set of experiments, a nonspecific mitogenic effect of the virus preparation was observed. This effect was obviously caused by soluble molecules contained in the virion-free supernatant obtained after ultracentrifugation of the viruses. Subsequently, we obtained highly purified virions which did not share the nonspecific mitogenic effect. The data obtained with these virions showed no responses of untreated control animals but a short-lived in vitro lymphoproliferative response 2 to 3 weeks after infection. This response then disappeared and was followed by a period of non-responsiveness which lasted as long as we tested the animals.

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.

Economic Forces Underlying Urban Decentralization Trends: A Structural Model for Density Gradients Applied to Korea

With the rapid pace of urbanization the structure of cities in developing countries is subjected to the very strong pressures of rapidly increasing population, rising incomes, and shifting factor prices. The standard procedure to document shifts in the structure of cities has been the use of density gradients. There is a quasi-universal tendency towards decentralization and the flattening of population-density gradients in all cities of the world. The shifting of density gradients is of great empirical importance to policymakers and we need to know more on how various economic and demographic forces operate to yield decentralization. Using a unique set of structural estimates for the housing and residential-land markets in Korea, this paper shows how an estimable structural model can be used to explore the specific economic forces affecting the shape of density gradients of Korean cities. Crucial estimates of the elasticity of substitution for housing in various Korean cities are presented. The indirect estimates of the shifts in the density gradients derived from the structural model are compared with the trends yielded by directly estimated density gradients. The implications of the analysis for our understanding of density gradients are drawn.

5] Isolation of microsomal subfractions by use of density gradients

This chapter provides information on the isolation of microsomal subfractions by use of density gradients. To avoid contamination of microsomes by fragments of organelles other than the endoplasmic reticulum (ER), relatively gentle homogenization (4 x 400 rpm) is required. Isolation of rough and smooth microsomes with a sucrose gradient containing cations is an effective and rapid procedure, but both cation-free water and careful maintenance of laboratory animals are required to avoid aggregation. It is possible to carry out density gradient centrifugation using metrizamide gradients because this substance has a low viscosity in aqueous solutions even when gradients with high density are formed. Metrizamide is nonionic, does not inhibit microsomal enzymes, and has no detergent effects on membranes, making this solute advantageous for density gradient centrifugation of microsomal fractions. It appears that deoxycholate liberates membrane fragments, which upon gradient centrifugation equilibrate in different bands. The presence of similar fragments in high concentration within the same band may result in aggregation of these fragments to larger particles.

SEPARATION OF RBCs WITH IMPROVED SURVIVAL FROM NORMAL BLOOD

This study investigated the use of density gradients to prepare RBCs with greater potential “in vivo” survival. Solutions of the desired density, osmolarity, salt composition and pH were easily prepared with Stractan II (S-11), an inexpensive water soluble arabino-galactan polymer of 30,000 daltons. RBCs were centrifuged on parallel discontinuous gradients of S-11 and crystalline albumin. Similar age dependent separation in both systems was indicated by the same rates of decline of reticulocytes, glucose-6-phosphate-dehydrogenase, pyruvate-kinase and hexokinase. Rabbit RBCs were divided on single layers of S-11 into light (young) and heavy (old) fractions of varying proportions. Either fraction or both or unfractionated RBCs were labelled with 51Cr alone or with 1251 also and reinjected into the donor. The rate of decline of radioactivity was followed until total disappearance. When compared to the unfractionated cells, the rate of decline of radioactivity was similar in the combined fractions; it was slowest and fastest in the light and heavy 10$ fraction respectively and it was significantly slower and faster in the light and heavy 50% fractions respectively. Corresponding differences were found for the interval to total disappearance thus ruling out differential label elution. These findings suggest that this technique may be used to obtain sizeable RBCs fractions of improved survival for transfusion to patients with chronic anemia.

Density-gradient centrifugation of mouse liver mitochondria with H 2O/D 2O gradients

The use of density gradients constructed with mixtures of H 2 O and D 2 O for sedimentation studies of mouse liver mitochondria is described. A major advantage of the technique is the avoidance of a solute gradient and the possibility of membrane damage. Distribution patterns of mouse liver mitochondria enzymes confirm the evidence for biochemical heterogeneity observed with sucrose gradients.

Histochemistry XLIV. Use of density gradient for isolation of mast cells.

SummaryThe procedure of Padawer and Gordon for the isolation of mast cells from peritoneal washings was modified to employ a sucrose density gradient, and along with other changes in certain details, a procedure was elaborated that regularly provides mast cells 100% pure with respect to cell type in yields up to 87% with respect to the original washings.